Case Studies in Cholera: Lessons in Medical History and Science

MEDICAL REVIEW

Stephen M. Kavica, Eric J. Frehmb, and Alan S. Segalc aDepartment of Surgery, Yale University School of Medicine, New Haven, Connecticut; bDepartment of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania; and CDepartments of Medicine, Molecular Physiology and Biophysics, and Pharmacology, University of Vermont, Burlington, Vermont

Cholera, a prototypical secretory diarrheal disease, is an ancient scourge that has both wrought great suffering and taught many valuable lessons, from basic sanitation to molecular signal transduction. Victims experience the voluminous loss of bicarbonate-rich isotonic saline at a rate that may lead to hypovolemic shock, metabolic acidosis, and death within afew hours. Intravenous solution therapy as we know it wasfirst developed in an attempt to provide life-saving volume replace- mentfor cholera patients. Breakthroughs in epithelial membrane transport physiology, such as the discovery ofsugar and salt cotransport, have paved the wayfor oral replacement therapy in areas of the world where intravenous replacement is not readily available. In addition, the discovery of the cholera toxin has yielded vital information about toxigenic infectious diseases, providing a framework in which to study fundamental elements of intracellular signal transduction pathways, such as G-proteins. Cholera may even shed light on the evolution and pathophysiology of cystic fibrosis, the most commonly inherited disease among Caucasians. The goal of this paper is to review, using case studies, some of the lessons learned from cholera throughout the ages, acknowledging those pioneers whose seminal work led to our understanding ofmany basic concepts in medical epidemiology, microbiology, physiology, and therapeuticsf

INTRODUCTION a short, curved, bacterium that produces an enterotoxin (choleragen). Humans are the Cholera is an infectious disease only known host for cholera vibrios [1], whose course can vary from a mild diar- which can spread rapidly though a com- rheal syndrome to a rapidly fatal malady munity primarily via fecal contamination (cholera gravis) characterized by the sud- of drinking water. den onset of profuse watery diarrhea with Throughout its history, cholera caused volume depletion, metabolic acidosis, and widespread panic because it can afflict an ultimately death from hypovolemic shock. otherwise healthy person without warning The disease is caused by Vibrio cholerae, and lead to death in as little as four to six c To whom all correspondence should be addressed: Alan S. Segal, M.D., University of Ver- mont, Ion Channel Facility, 208 South Park Drive, Suite 2, Colchester, VT 05446. Tel.: 802- 656-8916; Fax: 802-656-8914; E-mail: [email protected] d Abbreviations: Ace, accessory cholera enterotoxin; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CTX, cholera toxin; KATP, ATP-sensitive K+; ORT, oral rehydration therapy; tcpA, toxin regulated pili, Zot, zonula occludens toxin. Received: April 10, 1997; Accepted: March 28, 2000 393 394 Kavic et al.: Cholera: medical history and science

hours. Many victims have awoken with and had poor skin turgor. The wrinkled uncompromised health at the edge of the skin on her hands appeared as if it had day and died gruesomely in their own been immersed in water for a prolonged dejecta before night's retum. More recent- time ("washerwomen's hands"). Her facial ly in several parts of the world, cholera features were flattened and her eyes were pandemics have claimed thousands of sunk deep into their sockets. A radial pulse lives in outbreaks in southern Africa and was not palpable, but a heart rate of 160 Latin America. per minute was assessed by listening over We will present three case scenarios the chest. The patient's breathing was based on actual patients illustrating many labored, at a rate of 32 breaths per minute of the clinical manifestations of cholera. (hyperventilation in response to metabolic These will be used as a springboard to acidosis), but breath sounds over the lung review 1) the colorful history of cholera fields were clear. The abdomen was soft. including its impact on the origin of intra- The extremities were cold and there was venous solution therapy, 2) the fundamen- no peripheral edema. tal physiological principles that underlie Inspection of the canister showed the disease and its treatment, and 3) how approximately ten liters of nearly odorless aspects of cholera have intertwined with watery fluid containing flecks of mucus developments in modern science. ("rice-water" stools). Sensing the gravity of the patient's condition, the apothecary boiled eight gallons (30 liters) of water CHOLERA: SYMPTOMS, SIGNS, AND THE ORIGIN OF INTRA- and prepared two solutions: VENOUS THERAPY Solution #1: 5 gallons (-19 liters) of a solution containing by weight 9 parts sodi- Case Scenario 1: um chloride to 1000 parts water (0.9% iso- Sunderland, Britain, 1831 [2] tonic NaCl). Solution #2: 3 gallons (-11 liters) of a A 25-year-old woman was found in solution containing 12.6 parts soda of grave distress. The patient was too lethar- bicarbonate to 1000 parts water (isotonic gic to provide a history, but a friend states bicarbonate solution). that she was in her usual state of good As the patient was sinking towards health until 24 hours previously when she death, and as the bewildered townspeople developed the acute onset of profuse looked on, a hollow silver needle watery diarrhea, vomiting, and muscle (designed by John Read [3]) was placed in cramps. The villagers had collected all of the right femoral vein and attached to a her diarrheal fluid in a canister kept near breast pump syringe by a flexible tube the patient, since they believed that part of fashioned from goose trachea. A quart of the reason she was so ill was that her body the NaCl solution was injected over fifteen had been depleted of vital substances con- minutes, followed by a pint of the tained in the excretions. No other history NaHCO3 solution over the next fifteen was available except that several other vil- minutes. Three parts potassium chloride lagers had already died from this type of was also added to 1000 parts Solution #1 illness. The first death in Sunderland had (KCI 40 meq/l) in a few quarts. After five been on Oct. 26, 1831. cycles of this treatment, to the astonish- On physical examination, the patient ment of all who had gathered, the patient was extremely weak and lethargic. The became alert and animated. Her radial silver-bluish color of her skin was remi- pulse returned at a rate of 112 per minute. niscent of lead. She was cool to the touch The two solutions were continued, in Kavic et al.: Cholera: medical history and science 395 alternating fashion, until the patient stated 40 mg/dl. Two hours after admission, a that her bladder was full. By that time she second total CO2 level was only 5 meq/l. had received eight quarts of the NaCl solu- Central venous access was obtained and tion (four containing KCI) and eight pints intravenous lactated Ringer's solution of the NaHCO3 solution, a total of over infused. Supplemental potassium and eleven liters. bicarbonate was administered, along with The patient continued to pass "rice- intravenous gentamicin. After six hours, water" stools and required sixteen liters of the patient's condition improved signifi- intravenous solution therapy the next day cantly, with full return of peripheral pulses (Figure 1). By the fifth day, the diarrhea and intact mental status. had significantly decreased, but the patient The attending physician reviewed the had not passed any urine since day three. literature and found his patient's symp- Despite the valiant intravenous solution toms were compatible with cholera, replacement therapy, the patient died of althoiugh no cases of domestically progressive renal failure on day twenty [4]. acquired cholera had been reported in the Nevertheless, this was the birth of intra- United States since 1911 [6]. The physi- venous solution therapy as we know it. cian switched the antibiotic regimen to tetracycline and alerted the local health authorities. The diagnosis of cholera was CHOLERA IN THE UNITED STATES: confirmed by laboratory culture, which EFFECTIVE THERAPY identified Vibrio cholerae, El Tor biotype, Case Scenario 2: Inaba serotype. Further volume resuscita- Port Lavaca, Texas, 1973 [5] tion ensued, and the patient was dis- charged home in good health one week A 51-year-old man presented to the from initial presentation. emergency department with a four-hour history of profuse watery, non-bloody CHOLERA TODAY: VITAL ROLE OF diarrhea. The patient noted mild abdomi- REHYDRATION THERAPY nal pain along with non-odorous diarrhea, ORAL and experienced one episode of emesis. Case Scenario 3: The patient also complained of severe leg Dhaka, Bangladesh, 1999 [7] cramps. On initial physical examination, no A 29-year-old man presented to the blood pressure could be obtained. The clinic with a ten-hour history of diarrhea pulse was auscultated at 150 beats per and a seven-hour history of vomiting. The minute, and Kussmaul-type respirations patient could provide no further coherent were present at a rate of thirty breaths per history. On examination, the patient was in minute. The patient was afebrile, alert and mild distress, disoriented to place and time oriented. The abdominal findings were but talkative. The patient was afebrile, minimal - soft, scaphoid abdomen, non- tachycardic, and had a blood pressure of tender to palpation, with active bowel 90/60. The patient had sunken eyes and sounds. His extremities were noted to have poor skin turgor, but otherwise had an "splotchy" cyanosis, and no peripheral unremarkable exam. pulses were palpable. Based on local disease patterns, the Initial laboratory values included a patient was treated with the presumptive white blood cell count of 13,400 per ml, diagnosis of cholera, which was subse- hemoglobin of 18.1 g/dl, [Na] 136 meq/l, quently confirmed with darkfield illumina- [K] 3.1 meq/l, [CO2] 18 meq/l and BUN tion of the stool. The patient was given six 396 Kavic et al.: Cholera: medical history and science

18000 _ 16000 - * Urine (ml/day) 14000 7-o Stool (ml/day) 1-0/ - Intake (ml/day) 120000-

E 80000 0 6000- . Death

4000

2000

0 5 10 15 20 Hospital Day

Figure 1. Chart of the total input, total output, and stool output for the patient described in case scenario one. Note that stool accounts for nearly all the output throughout the hospital course, and that initial stool output approached ten liters per day. The patient died of oliguric renal failure on hospital day 20.

hours of intravenous therapy with Dhaka osmolarity of 31 1 mosmll. The patient was solution [6]: [Na] 133 mmol/l, [K] 13 given a volume of WHO-OHS equivalent mmol/l, [Cl] 98 mmol/l, and [acetate] 48 to the total stool/emesis output, collected mmol/1), for a total volume of six liters. in graduated containers beneath a cholera This solution provides the volume, potas- cot. The patient was also given free access sium, and bicarbonate (as acetate is con- to food and water. verted to bicarbonate in the liver) required The total stool output in the first day to offset the massive losses of these elec- was over 12 1, but the total output over the trolytes in the diarrhea of cholera. Ery- next two days was 4.21. The patient's diar- thromycin therapy was also instituted, 500 rhea completely resolved 48 hours after mg every six hours for three days. presentation. The patient consumed a total At the completion of the initial resus- of twenty-one liters of WHO-OHS and an citation, the patient was coherent and additional two liters of free water. Final showed no signs of systemic infection. laboratory analysis confirmed infection The intravenous fluids were discontinued. with Vibrio cholerae, El Tor Ogawa. The patient was given World Health Orga- nization Oral Rehydration Solution (WHO-OHS), containing [Na] 90 mmol/l, THE HISTORY OF CHOLERA [K] 20 mmol/l, [Cl] 80 mmol/l, [citrate] 10 Cholera has been endemic to the Indi- mmol/l (which is metabolized to bicarbon- an subcontinent for millennia. Spread of ate), and [glucose] 111Immol/l (to facili- cholera beyond the Indian subcontinent in tate Na-glucose cotransport), for a total 1817 marked the first worldwide pandemic Kavic et al.: Cholera: medical history and science 397

(see Table 1). However, compelling came in 1883 when Robert Koch, discov- descriptions of severe diarrheal diseases erer of the etiologic agent of tuberculosis, consistent with cholera dating from the led an expedition to Egypt during which time of Hippocrates suggest the possibility he identified a microbe he called komma- of an even earlier emergence from Asia [8]. bazillen (comma bacilli) [11]. He subse- In 1959, Robert Pollitzer chronicled quently detected kommabazillen in the the first six pandemics [9]. Although reli- stools of cholera patients in Calcutta. The able serotyping was not available prior to name of this bacterium was later changed the fifth pandemic, one might presume that to Vibrio cholerae, in belated recognition the first four pandemics were caused by of Filipo Pacini, who had bestowed the lat- the same agent (Table 1). The first case ter name on the bacillus he observed in the presented was based on cholera's first excreta of cholera victims in Italy thirty arrival in Britain during the second pan- years earlier [9]. demic. Vibrio cholerae 01 has two vari- Fortunately, even before the etiologic ants, the so-called classical biotype; and agent was identified, there were those com- the El Tor biotype, the hardier but less vir- mitted to the notion that the spread of ulent agent of the seventh pandemic. Since cholera could be controlled ". . . not with 1992, outbreaks of cholera caused by a prayer and fasting, but through disinfection new serogroup, Vibrio cholerae 0139, and quarantine. [C]holera demonstrated have been reported in South Asia and forcefully that a disease that could not be China, raising the prospect of an Eighth cured must be prevented" [12]. From con- Pandemic [10] (Table 1). victions such as these grew institutions like In 1854, John Snow linked a cholera New York City's Metropolitan Board of outbreak in London to a single source: a Health, credited with containing a cholera contaminated water pump on Broad Street. outbreak in 1866. The association of This was the first convincing demonstra- cholera with unsanitary water and crowd- tion that cholera was a contagious and ed, squalid living conditions has always water-borne disease. The removal of the resulted in a disproportionate representa- pump handle at Dr. Snow's request stands tion of the urban poor on its death rolls. as a landmark in the history of public Perhaps it was inevitable that so dreadful a health and preventive medicine. Another scourge - one that generally spared the milestone in the understanding of cholera wealthy and refined while ravaging the

Table 1. The Cholera Pandemics (based on Pollitzer, 1959 [8]). Pandemic Time Period Serogroup Biotype Affected Areasb First 1817to 1823 VibriocholeraeOl1 Classicala 1, 2,3,5 Second 1829 to 1851 Vibrio cholerae 01 Classicala 1, 2, 3, 4, 5,6, 7 Third 1852 to 1859 Vibrio cholerae 01 Classicala 1, 2, 3, 4, 5, 6, 7, 8 Fourth 1863 to 1879 Vibrio cholerae 01 Classicala 1, 2, 3, 4, 5, 6, 7 Fifth 1881 to 1896 Vibrio cholerae 01 Classical 1, 2, 3, 4, 5, 6, 7 Sixth 1899 to 1923 Vibrio cholerae 01 Classical 1, 2, 3, 4, 5, 7 Seventh 1961 to Present Vibrio cholerae 01 El Tor 1, 2, 3, 4, 5, 8, 9 Eighth (?) 1992 to Present Vibrio cholerae 0139 Single biotype 2 a Presumed. b Affected areas: 1, Indian Subcontinent; 2, Southeast Asia; 3, Middle East; 4, Europe; 5, East Africa; 6, North Africa; 7, North American; 8, South America; 9, Indonesia. 398 Kavic et aL: Cholera: medical history and science

impoverished and uneducated - would be day, the 500 ml absorbed represents about seen as divine retribution for the sinful, 10 percent of the colon's absorptive capac- profligate, and unworthy. Indeed, for prop- ity. er members of the upper class, Vibrio Fluid transport in the small intestine cholerae could be both an intestinal generally results from the passive move- pathogen and a character assassin - "to ment of water across epithelial membranes die of cholera was to die in suspicious cir- driven by osmotic and hydrostatic pres- cumstances" [13]. sures. In the absence of food, ions are the Over a century later, can we be sure most important contributors to osmotic that a parallel situation does not exist for pressure in the lumen. Since the cells of patients with AIDS, who may face similar the small intestine are relatively permeable injustices as a consequence of societal (leaky), luminal fluid generally remains ignorance? Long before this terrible dis- isotonic with plasma. Because most water ease challenged today's scientists explor- absorption occurs in the absence of a ing the intricacies of cellular signaling and transmucosal osmotic gradient, any toxin-mediated disease, cholera chal- increase in the osmotic content of the lenged the very idea of what a "disease" lumen will tend to reduce water absorption actually was, what it was not, and what and lead to diarrhea. Since 60 to 90 per- thinking citizens might do about it. cent of stool weight is water, diarrhea is mainly due to excess fecal water. Sodium: Sodium is actively absorbed NORMAL INTESTINAL PHYSIOLOGY throughout the small intestine, the site most affected in cholera. The favorable Under normal circumstances, the electrochemical gradient (negative mem- human gastrointestinal tract can absorb brane potential and low intracellular [Na]) more than 90 percent of the water and ions allows sodium to enter the cell across the presented to it. To appreciate fully the apical brush border, and exit across the pathophysiology of cholera, a brief review basolateral membrane via the Na, K of normal water and solute handling by the ATPase pump. The net rate of Na+ absorp- intestines is helpful. tion is highest in the jejunum, where it is Water: The healthy small intestine is enhanced by the presence of glucose, able to absorb about 7 - 8.5 liters of the galactose, and neutral amino acids via nine liters that enters it daily, leaving an Na+-sugar cotransport. Indeed, the identi- ileocecal flow rate of 0.5-2 Il/day. It is fication of Na+-coupled cotransport sys- important to remember that in the small tems stands as one of the most important intestine, mature epithelial cells near the medical discoveries of this century, since tips of the villi are engaged in net absorp- these processes form the basis of oral tion, while the less mature cells in rehydration therapy used in many parts of Lieberkiihn's crypts function in the net the world. The net rate of Na+ absorption secretion of water and electrolytes. Typi- is smaller in the ileum due to a lower den- cally, only about 600 ml of fluid is pre- sity of cotransporter proteins. sented to the colon per day, of which 500 Potassium: The movement of potassi- ml are absorbed, leaving the 100 ml con- um in the small intestine is usually in the tained in the average daily fecal weight of direction of absorption. As water is 150 g. Therefore, one functional definition absorbed luminal [K] rises, thereby of diarrhea is an average daily fecal weight increasing the driving force for absorption. exceeding 200 g. Since the colon is capa- It is important to note that since most K+ ble of absorbing 4 to 6 liters of water per absorption results from its rising luminal Kavic et al.: Cholera: medical history and science 399

concentration as water is absorbed, signif- rather the patient becomes severely vol- icant K+ loss may occur in diarrhea, espe- ume-depleted. Conceptually, it is absolute- cially in the high-flow secretory diarrhea ly critical to distinguish hypovolemia (loss of cholera. of salt in isotonic proportions) and dehy- Anions: The jejunum absorbs large dration (loss of solute-free water) [14]. amounts of both Cl- and HCO3- anions, Hypovolemia is assessed clinically by and by the end of the jejunum most of the physical exam and does not require any HC03- from hepatic and pancreatic secre- laboratory tests, and the diagnosis of salt tions has been absorbed. It follows that (volume) depletion implies the life-saving diseases affecting the small intestine can therapy of isotonic salt (volume) repletion. result in significant HC03- losses. In contrast, "dehydration" implies solute- free water loss, or hypertonicity, which is a laboratory diagnosis. PATHOPHYSIOLOGY OF CHOLERA Chloride secretion Cholera stimulates solute and water secretion in the small intestine. With con- The transport function of epithelia is tinuous parenteral fluid replacement, the inextricably linked to the polarized distrib- ileocecal flow rate may reach 15 to 20 ution of transport elements in the apical liters per day. Such a secretory diarrheal and basolateral membranes of epithelial process can lead to severe losses of vol- cells (Figure 2) [15]. Secretion of fluid ume and electrolytes. The initial stool out- across the intestinal epithelium is primari- put in cholera gravis can exceed a full liter ly driven by the net transport of solute into per hour, resulting in death in as little as the lumen and the secondary movement of four to six hours. It has been said that water down the resultant osmotic gradient. cholera is a disease that begins where Net transport in a preferred direction other diseases end - with death. requires an asymmetric arrangement of It is important to emphasize that since transport elements; that is, cell polarity. the fluid loss in cholera is essentially iso- The transepithelial secretion of chlo- tonic, "dehydration" is not the problem; ride is central to fluid secretion across

2 K+ 3 Na+ Apical Basolateral lumen K+ - membrane

+CI -- Cl <7X Na-'Na+~~~Cl CFTR K+

Figure 2. Secretory epithelial cell model. The Na, K-ATPase pump and triple ion trans- porter are shown on the basolateral side of the cell, with the CFTR chloride channel illus- trated on the apical membrane. This arrangement allow for secretion of chloride across the cell. 400 Kavic et al.: Cholera: medical history and science many secretory epithelia including the From such considerations, it follows small intestine. In secretory epithelial cells that transcellular C1- transport is influ- such as those in the crypts of Lieberkuhn, enced by the electrochemical (Nernst) the asymmetry of Cl- transport elements potential for K+. Assuming typical values underlies Cl- secretion. The basolateral for intracellular and extracellular [K] of Na,K-ATPase pump drives active Cl- 120 mM and 4.4 mM, respectively, the transport, and inhibitors of the pump such Nernst potential for potassium (EK) would as ouabain abolish Cl- secretion. The low be -84 mV. However, for the hypokalemia intracellular [Na] maintained by the pump existing in a patient ([K] = 3.2 mM) EK and the low intracellular [Cl-] favor the increases to -92 mV, an 8 mV hyperpolar- entry of Cl- across the basolateral mem- ization. Under these conditions, as baso- brane via a furosemide- and bumetanide- lateral K+ channels open to allow K+ to sensitive Na:K:2Cl triple ion cotrans- recycle, the membrane potential hyperpo- porter. The pump also maintains a high larizes' even more than with normal potas- intracellular [K], and together with the sium levels, which heightens the increase large basolateral K+ conductance, causes in the driving force for chloride secretion the apical and basolateral membrane when apical CFTR Cl- channels open. potentials to be cell-negative. This nega- Even if EK were to remain constant, tive membrane potential provides a key an increase in the basolateral conductance driving force for conductive Cl- exit when for K+ would result in hyperpolarization of chloride channels localized in the apical the cell membrane potential. The presence membrane open. of ATP-sensitive K (KArp) channels in the Thus, the active secretion of Cl- by basolateral membrane could provide such polarized epithelia is mediated by an a mechanism [18], since an increase in asymmetric arrangement of a basolateral transport activity requires an increased Cl- entry element (the electroneutral triple turnover of the Na,K-ATPase pump, which cotransporter) and an apical Cl- exit ele- tends to lower intracellular ATP levels. ment (a transmembrane channel). Simply Therefore, the development of put, transcellular transport requires a way hypokalemia enhances the secretion of C1- in and a way out. Of great significance is into the lumen. This addition of Cl- to the that the major Cl- conductance in the api- lumen then provides both an electrical dri- cal membrane of secretory epithelia ving force for the paracellular movement (including the crypt cells) is the now of Na+ and an osmotic driving force for famous Cystic Fibrosis Transmembrane water to go into the lumen. The driving conductance Regulator (CFTR)d, the pro- force for water flow into the lumen sub- tein that is defective in patients with cystic sists when the luminal fluid becomes iso- fibrosis (CF). CFTR is a multi-functional osmotic, which explains why the secretory protein, but its characterization as a diarrhea of cholera is essentially isotonic. cAMP-activated Cl- channel is well docu- To make matters worse, the increasing mented [16, 17]. In secretory epithelia, water content of the lumen "dilutes" the continued Cl- secretion hinges on the luminal [K], reducing the major driving basolateral membrane being selectively force for K+ reabsorption, promoting fur- conductive to K+, while CFTR in the api- ther K+ loss. The process then becomes a cal membrane is selectively conductive to vicious cycle of isotonic Na+ (with Cl- and C1-. Basolateral K+ efflux hyperpolarizes HCOj) and K+ loss, resulting in progres- the cell membrane potential, which sive hypovolemia, hypokalemia, and meta- increases the driving force for Cl- secre- bolic acidosis. Without intervention, the tion. Kavic et al.: Cholera: medical history and science 401 patient dies of hypoperfusion acidosis and The precise mechanism of action is hypovolemic shock. still under investigation, but several stud- Cholera toxins ies have yielded information on both its structure [22, 23] and action [24, 25, 26]. It is not an overstatement to say that There seems to be a differential effect of the entire clinical syndrome of cholera is the toxin on the enterocytes: it exerts a due to the action of the cholera toxin on direct stimulatory effect on the secretory intestinal epithelial cells. Vibrio cholerae crypt cells, and an anti-absorptive effect is a non-invasive, aerobic, gram-negative on the villous cells, which both favor net bacillus that produces the choleragen, the secretion. Fortunately, the decrease in cholera toxin (CTX). The organism attach- absorption is much less than the increase es to the intestinal mucosa via various sur- in secretion, making oral rehydration ther- face adhesion components, such as toxin apy (ORT) possible (vida infra). The colon coregulated pili (tcpA) [19]. The most sen- is usually in a state of absorption since it is sitive regions are in the duodenum and relatively insensitive to the toxin, but its upper jejunum; the ileum is less affected. absorptive capacity is quickly overcome in The organism does not invade the mucosal this "overflow" diarrhea. surface, and bacteremia is virtually never The crystal structure of CTX was seen. CTX is cytotonic rather than cyto- recently solved [22], and it closely resem- toxic, as it disrupts intracellular processes bles the heat-labile enterotoxin from but does not directly cause cell death. Escherichia coli, LT [27]. Both are het- Although Koch identified Vibrio erotrimeric complexes, comprised of a cholerae as the etiologic agent of cholera, pentameric arrangement of five B subunits and first proposed that the disease was within which the wedge-shaped A subunit toxin-mediated in 1884 [10], CTX has is loosely held [28]. Choleragenoid, the B only been characterized in the latter half of subunit pentamer of cholera toxin, directs this century. In 1959, De showed that inoc- the enzymatic A subunit to its target by ulation of ligated segments of rabbit small binding to the GM1 gangliosides exposed intestine with the sterile and bacteria-free on the luminal surface of intestinal epithe- filtrate from cultures of Vibrio cholerae lial cells [29, 30, 31]. Interestingly, the two caused marked distension due to the secre- enterotoxins share approximately 80 per- tion of isotonic fluid [20]. No distension cent sequence homology, but the other 20 developed in the ligated segments from the percent may be the difference between a same rabbits injected with control medi- self-limited disease and death. The most um. This was the first convincing demon- significant difference between the two tox- stration that Vibrio cholerae must be elab- ins is the carboxyl terminus of the A2 orating a soluble factor that itself is capa- chain, which helps to tether the A subunit ble of mediating the disease. above the plane defined by the five B sub- The toxin satisfies all of "Koch's pos- units. Apparently, it is differences such as tulates," since the toxin: (1) is present in these that lead to the production of much those affected and absent in healthy indi- higher levels of cAMP with CTX than viduals, (2) can be isolated from infected with LT. patients (in this case, from their stool) and The A subunit is the active component produced in culture, (3) is able to cause of the toxin [26]. It is thought that the disease when reinoculated into susceptible entire A subunit (or a portion of it) gains hosts from pure culture, and (4) can be re- entry to the cell through the ring of the B isolated from the experimentally infected subunits on the apical membrane [32]. The host [21]. Al peptide, the active fragment of the A 402 Kavic et al.: Cholera: medical history and science subunit cleaved by proteolysis, binds to ciation of the Py complex from the a-sub- NAD inside the cell [33, 34]. The toxin unit [45]. Finally, the ADP-ribosylation by subunit then catalyzes the addition of an cholera toxin prevents the dissociation of ADP-ribose from NAD to the G protein the a-subunit from the activated cyclase, that activates adenylyl cyclase, Gs [17, 35, keeping the enzyme locked in its active 36]. The cAMP produced activates the state [42, 46]. cAMP-dependent protein kinase (PKA), The ADP-ribosylation by cholera whose catalytic subunit then phosphory- toxin requires the presence of separate lates apical membrane chloride channels. ADP-ribosylation factors, or ARFs [47, There is some evidence that other proteins 48]. ARFs are GTP-binding proteins, nor- are also ADP-ribosylated, but the signifi- mally present in cells, that function in con- cance of these other additions has not yet junction with the cholera toxin to increase been determined [37]. the level of adenylyl cyclase activity [49]. Normal physiology of G-proteins ARFs appear to be allosteric activators of the Al peptide, interacting directly with The signal transduction mechanism the toxin to stimulate its activity in vivo involves heterotrimeric guanine nucleotide [50, 51, 52]. Currently three highly con- binding (G) proteins comprised of an a served classes of these factors have been subunit, and a Py complex (see reviews identified, and different ARFs may be have [38, 39, 40]). Typically, a first messenger optimal activity under different environ- (e.g., a hormone) binds to a membrane mental conditions [53, 54]. receptor, activating a G protein within the Thus, the result of the action of membrane. The a-subunit of the activated cholera toxin is an elevation of cAMP to G protein then exchanges GTP for GDP pathologically high intracellular levels and dissociates from the fry complex. This through a G protein mediator. CTX- activated a-subunit directly interacts with induced elevations in cAMP lead to an an effector molecule or enzyme such as abnormally high apical membrane chlo- adenylyl cyclase. ride conductance, due to opening of the One of the remarkable features of the CFTR chloride channels. Chloride can a-subunit is its intrinsic GTPase enzymat- then move down its concentration gradi- ic activity, providing an auto-negative ent, spilling from the cell in abnormally feedback mechanism for regulated deacti- high quantities. Due to the resultant elec- vation [41]. Once the GTP is hydrolyzed tronegative lumen, sodium is secreted via to GDP, the a-subunit may re-associate a paracellular pathway. This osmotic gra- with the fy complex, thereby turning off dient drives water flow into the lumen, the functional activity of the effector mol- with the net effect being the secretion of ecule. massive amounts of isotonic fluid. toxin Proteins Cholera toxin exerts additional effects, Cholera and G separate from the adenylyl cyclase system. Cholera toxin activates adenylyl For instance, CTX also triggers intestinal cyclase in at least three ways through the enterochromaffin cells to release serotonin ADP-ribosylation of the a-subunit [23, [55]. Serotonin may contribute to height- 42]. First, ADP-ribosylation reduces the ened gastrointestinal secretion by serving inherent GTPase activity of the a-subunit as a excitatory neurotransmitter of intra- by a factor of one hundred, resulting in a mural neurons within the enteric nervous tremendous increase in the production of system [56, 57]. VIP-containing neurons cAMP [43, 44]. Second, the addition of may serve as the effectors of vasodilata- ADP-ribose facilitates the physical disso- Kavic et al.: Cholera: medical history and science 403 tion, increasing mucosal perfusion and, as maintains the frequency of this otherwise a result, intestinal secretion [58, 59]. deleterious recessive gene in the general Furthermore, Vibrio cholerae pro- population. duces other toxins in addition to CTX A similar advantage has been hypoth- [60]. Zonula occludens toxin (Zot) esized for the diseases of cholera and cys- increases intestinal permeability by weak- tic fibrosis (CF) [66, 67, 68]. In CF, the ening the tight junctions that maintain the apical chloride channel (CFTR) is hyper- integrity of the epithelial sheet [61]. Zot active or inactive, preventing the direct interacts with specific cell receptors, transport of chloride across cellular mem- reversibly affecting the actin elements of branes [69, 70]. This homozygous reces- the cytoskeleton, allowing increased loss sive disorder occurs at a frequency of one of ions into the intestinal lumen [62]. in 2500 Caucasian live births, an alarm- Accessory cholera enterotoxin (Ace) also ingly high rate given the severity of the contributes to secretory diarrhea, possibly disease [71]. This high rate is inadequate- by opening channels and increasing the ly explained by the effects of genetic potential difference across the apical mutation or by a fertility difference of the membrane [63]. Pathogenic strains of Vib- carriers [72, 73]. rio cholerae may also produce a sodium Heterozygotes for CF have some channel inhibitor [64]. These additional abnormal channels due to the mutant gene, toxins have a supplementary role to CTX as well as normal channels due to the wild- in the pathogenesis of secretory diarrhea in type gene. In the absence of infection or cholera. severe stressors, the heterozygote would suffer few clinically evident abnormalities, due to the presence of the subset of nor- CHOLERA AND THE HETEROZYGOTE ADVANTAGE mally functioning channels [74]. However, HYPOTHESIS it is possible that when heterozygotes for CF become infected with Vibrio cholerae, The impact of cholera goes well the increase in apical chloride conductance beyond the clinical syndrome of cholera is blunted, thereby attenuating the massive gravis. To the present day, cholera contin- loss of salt associated with those cholera ues to challenge definitions of disease and victims having a full complement of nor- the underlying mechanisms that maintain mal CFTR channels [75, 76]. A recent diseases within the human population. On experiment confirmed this hypothesis a fundamental level, the most important using transgenic mice, showing that chlo- question is why certain diseases (like cys- ride secretion was directly correlated to tic fibrosis) exist at all, and to answer it, the number of CFTR alleles [77]. Howev- we are again confronted with the problem er, other experiments have not provided as of cholera. conclusive data [78]. In select circumstances, there can be a Cholera has remained a scourge theoretical advantage to heterozygosity for throughout the latter half of the last centu- a damaging trait, when this combination of ry. Considering the time scale necessary alleles protects the carrier from another for adaptation, not enough time has passed severe disorder. In perhaps the best known to remove the allele from the gene pool example, heterozygotes for the abnormal [2]. Thus, it is possible that CF continues protein Hemoglobin S do not have sickle to exist in the human population because cell anemia, but are protected to a certain heterozygosity for this disorder is to some degree against malaria [65]. Thus, there extent protective against cholera. exists a "heterozygote advantage" that 404 Kavic et al.: Cholera: medical history and science

TREATMENT at restoring balance to a hyperactive secre- Death from cholera gravis results tory system can be lifesaving. from untreated hypovolemic shock with To stop the losses in cholera, the metabolic acidosis. The cornerstones of source of the infection must be eliminated. therapy are 1) to give the patient back what Fortunately, the Vibrio infection itself is is lost - a lot of isotonic fluid - as the self-limited; that is, bacteria do not remain villagers believed; and 2) to stop the loss- active in the intestinal tract for extended es. To replete the lost substances in 1831, periods of time. Although antibiotics may physicians attempted intravenous volume decrease the severity of the clinical symp- resuscitation. However, if they would have toms or otherwise diminish the course of known about Na-coupled glucose trans- the illness, in most cases they are not port, they could have made an oral rehy- essential to the treatment of the disorder. drating solution containing NaCl, KCl, Therefore, in developing countries or in and bicarbonate of soda of the same com- those in which medicine is in short supply, position as what the patient was losing, a regimen of ORS will permit the survival along with glucose. of the vast majority of those infected [85], For this reason alone, the identifica- as in case scenario [3]. tion of sodium-coupled epithelial cotrans- In the present day, most physicians port processes was a major medical break- would begin treatment with intravenous through. Although Reid had provided isotonic saline, bicarbonate replacement, experimental evidence supporting the and antibiotics (tetracycline, doxycycline, transport of sugars against a concentration or ciprofloxacin [86]). It should be difference at the turn of the century [79], stressed, however, that ORS would and the dependence on luminal sodium was does work. ORS is particularly valuable in first observed by Riklis and Quastel in the countries where cholera is endemic and late 1950s [80]. In the early 1960s, Crane can be pandemic, as recently seen in Peru and colleagues advanced these ideas and in 1991. showed that sodium-dependent sugar cotransport occurred in the luminal brush border of small intestinal epithelial cells SUMMARY [81, 82]. The first of the family of sodium- Cholera has been a scourge of glucose transporters was subsequently humankind, claiming millions of lives cloned from rabbit small intestine by from antiquity to the present day. The his- Wright and coworkers in 1987 [83]. tory of cholera is also associated with the Normally the sodium-coupled trans- development of several medical break- porters utilize the large electrochemical throughs (e.g., intravenous solution thera- gradient of sodium to drive absorption of py, basic sanitation, oral rehydration ther- sugars and other nutrients (e.g., amino apy) that have saved millions of lives. acids). Although cholera toxin biases the Study of the cholera toxin continues to system strongly in the direction of secre- provide great insights into the cellular tion, reabsorptive processes are largely left basis of diseases, the molecular mecha- intact. In fact, one study involving total nisms of signal transduction systems intestinal perfusion with cholera toxin in involving G-proteins, and our understand- human volunteers demonstrated a dou- ing of epithelial transport. Such progress bling of net sodium cotransport after expo- has rendered cholera a curable disease. sure to CT [84]. Therapy with ORS takes Despite this level of advancement, it is advantage of cotransport, by using sugar to paradoxical that the fate of people with drive the reabsorption of salt. This attempt cholera in underdeveloped parts of the Kavic et al.: Cholera: medical history and science 405

J.C., and Gangarosa, E.J. A case of cholera world is no different from that of the in Texas, 1973. Am. J. Epidemiol. 100:487- unfortunate patient in 1831. 498, 1974. 6. Blake, P.A., Allegra, D.T., Snyder, J.D., Barrett, T.J., McFarland, L., Caraway, C.T., EPILOGUE Feeley, J.C., Craig, J.P., Lee, J.V., Puhr, N.D., and Feldman, R.A. Cholera - a pos- In 1831, intravenous therapy was not sible endemic focus in the United States. N. available. Indeed, it was during the cholera Engl. J. Med. 302:305-309, 1980. epidemic in Sunderland in 1831 that the 7. Alam, N.H., Majumder, R.N., Fuchs, G.J., and the CHOICE study group. Efficacy and idea of intravenous therapy was proposed safety of oral rehydration solution with by Dr. William Brooke O'Shaughnessy, a reduced osmolarity in adults with cholera:a 22-year-old graduate of Edinburgh, in a randomised double-blind clinical trial. paper on cholera to the Westminster Med- Lancet 354:296-299, 1999. 8. Barua, D. History of Cholera. In: Barua, D, ical Society. This wasn't attempted until and Greenough III, W.B, eds. Cholera. 1832 when Dr. Thomas Latta "dissolved New York: Plenum Medical Book Co.; from two to three drachms of muriate of 1992, pp. 1-24. 9. Pollitzer, R. Cholera. WHO, Geneva. 1959. soda and two scruples of the subcarbonate 10. Kaper, J.B., Morris, Jr., J.G., and Levine, of soda in six pints of water, and injected M.M. Cholera. Clin. Micro. Rev. 8:48-86, it at temperature 1120 Fah." (The Lancet of 1995. June 2, 1832). In modem units, this is 11. Koch, R. An address on cholera and its bacillus. Brit. Med. J. 2:403-407, 1884. about [Na] 58 meq/l, [Cl] 49 meq/l, and 12. Rosenberg, C.E. The Cholera Years: The [HCO3-i 9 meq/l [87]. Although they United States in 1832, 1849, 1866. Chica- didn't have antibiotics, they did have salts go: University of Chicago Press; 1962, pp. and sugars, and thus an oral rehydration 213-214. 13. Rosenberg, C.E. The Cholera Years: The solution would have worked. United States in 1832, 1849, 1866. Chica- go: University of Chicago Press; 1962, p. ACKNOWLEDGEMENT: This article was 42. based on work performed in Dr. Alan Segal's 14. Segal, A.S. Salty language is confusing. section ofthefirst yearphysiology course at the Hosp. Pract. 31:81-84, 1996. Yale University School ofMedicine, where Drs. 15. Segal, A.S., Boulpaep, E.L. and Mauns- Kavic and Frehm were his dedicated, if not bach, A.B. A novel preparation of dissoci- speedy, students. ated renal proximal tubule cells that maintain epithelial polarity in suspension. Am. REFERENCES J. Physiol. 270:C1843-C1863, 1996. 1. Rabbani, G.H., and Greenough III, W.B. 16. Welsh, M.J., Anderson, M. P., Rich, D.P., Cholera. In: Lebenthal, E, and Duffey, M, Berger, H.A., Denning, G.M., Ostedgaard, eds. Textbook of Secretory Diarrhea. New L.S., Sheppard, D.N., Cheng, S.H., Grego- York: Raven Press; 1990, p. 245. ry, R.J., and Smith, A.E. Cystic fibrosis 2. Benyajati, C., Keoplug, M., Beisel, W.R., transmembrane conductance regulator: a Gangrosa, E.J., Sprinz, H., and Sitprija, V. chloride channel with novel regulation. Acute renal failure in asiatic cholera: clini- Neuron 8:821-829, 1992. copathologic correlations with acute tubu- 17. Gadsby, D.C., Nagel, G., and Hwang, T.C. lar necrosis and hypokalemic nephropathy. The CFIR chloride channel of mammalian Ann. Intern. Med. 52:960-975, 1960. heart. Ann. Rev. Physiol. 57:387-416, 3. Astrup, P., Bie, P., and Engell, H.C. In: Salt 1995. and Water in Culture and Medicine. 18. Mauerer, U.R., Boulpaep, E.L. and Segal, Munksgaard Intl. Publishers, Copenhagen; A.S. Regulation of an inwardly rectifying 1993, p. 169. ATP-sensitive K+ channel in the basolater- 4. In a desperate effort to save the patient, the al membrane of renal proximal tubule. J. woman's abdomen was impaled with Gen. Physiol. 111: 161-180, 1998. another segment of goose trachea to initiate 19. Taylor, R.K., Miller, V.L., Furlong, D.B., peritoneal dialysis on day fifteen, but to no and Mikalanos, J.J. Use of phoA gene avail. fusion to identify a pilus colonization factor 5. Weissman, J.B., DeWitt, W.E., Thompson, coordinately regulated with cholera toxin. J., Muchnick, C.N., Portnoy, B.L., Feeley, Proc. Natl. Acad. Sci. 84:2833-2837, 1987. 406 Kavic et al.: Cholera: medical history and science

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