Vibrio cholerae Infection of Drosophila melanogaster Mimics the Human Disease Cholera

Cholera, the pandemic diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Cholera toxin, which is responsible for the voluminous stools of cholera, causes constitutive activation of adenylyl cyclase, resulting in the export of ions into the intestinal lumen. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods including insects. Here we report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease: (i) death of the fly is dependent on the presence of cholera toxin and is preceded by rapid weight loss; (ii) flies harboring mutant alleles of either adenylyl cyclase, Gsα, or the Gardos K+ channel homolog SK are resistant to V. cholerae infection; and (iii) ingestion of a K+ channel blocker along with V. cholerae protects wild-type flies against death. In mammals, ingestion of as little as 25 μg of cholera toxin results in massive diarrhea. In contrast, we found that ingestion of cholera toxin was not lethal to the fly. However, when cholera toxin was co-administered with a pathogenic strain of V. cholerae carrying a chromosomal deletion of the genes encoding cholera toxin, death of the fly ensued. These findings suggest that additional virulence factors are required for intoxication of the fly that may not be essential for intoxication of mammals. Furthermore, we demonstrate for the first time the mechanism of action of cholera toxin in a whole organism and the utility of D. melanogaster as an accurate, inexpensive model for elucidation of host susceptibility to cholera.


Introduction
Cholera continues to be a major cause of morbidity and mortality in many parts of the world [1]. It is contracted through ingestion of contaminated food or water and is characterized by profuse diarrhea and vomiting. Cholera toxin, the primary determinant of this clinical syndrome, is an AB 5 -type exotoxin composed of an A subunit noncovalently bound to five B subunits, arranged in a rosette to form a lectin recognizing the GM 1 ganglioside [2]. The mechanism by which cholera toxin enters intestinal epithelial cells and disrupts function has been studied extensively in cultured cells [3][4][5][6][7]. Prior to entry into the cell, the A subunit is proteolytically cleaved into a catalytic A 1 subunit and an A 2 subunit, whose role is to maintain the non-covalent association to the B subunit GM 1 lectin. This lectin forms an association with GM 1 gangliosides that are concentrated in lipid rafts within the cell membrane. Once bound to GM 1 , retrograde transport on lipid rafts delivers cholera toxin to the endoplasmic reticulum. The A 1 subunit then dissociates from the toxin complex and exits the endoplasmic reticulum to ADP-ribosylate the stimulatory G protein subunit, G sa . The modified G sa constitutively activates adenylyl cyclase, and levels of cAMP in intestinal epithelial cells rise. The consequent secretory diarrhea depends on opening of cAMP-responsive Cl À channels and flow of Cl À and water through the apical surface of the epithelial cell into the intestinal lumen. KCNN4, an intermediate conductance Ca 2þactivated K þ channel of mammals, maintains K þ export through the basolateral aspect of the intestinal epithelial cell. Clotrimazole, which blocks the KCNN4 channel, has been shown to decrease cholera toxin-induced Cl À secretion in both cultured mammalian cells and mice [8,9]. These results suggest that simultaneous basolateral export of K þ is required to maintain passage of Cl À through basolateral K þ /Cl À cotransporters and apical Cl À channels into the intestinal lumen.
The utility of Drosophila melanogaster as a model host for human pathogens is well-established [10][11][12][13][14][15][16][17][18]. In the natural environment, Vibrio cholerae is closely associated with arthropods [19][20][21], and many have suggested that insects serve as vectors [22][23][24][25][26] or reservoirs [27][28][29] of V. cholerae. Thus, we hypothesized that insects or related arthropods might serve as excellent model hosts of V. cholerae. To test this, we subjected the model insect D. melanogaster to oral V. cholerae infection. Here we demonstrate that V. cholerae infection of D. melanogaster exhibits the following parallels to human disease: (i) ingestion of V. cholerae produces an intestinally-localized, lethal infection in the fly that is dependent on cholera toxin; (ii) host susceptibility is dependent on Gsa, adenylyl cyclase, and the Drosophila KCNN4 channel homolog; and (iii) clotrimazole, an inhibitor of the human KCNN4 channel, protects the fly against infection. However, we have also found differences between V. cholerae infection of mammals and flies. Ingestion of cholera toxin alone is sufficient to cause severe secretory diarrhea in humans and model mammals [30][31][32][33]. In contrast, in the fly, we have found that ingestion of cholera toxin is lethal only when pathogenic isolates of V. cholerae are ingested in tandem. Our findings not only demonstrate the utility of the fly as a model host for V. cholerae infection, but also suggest that the V. cholerae genome contains virulence factors specifically required for infection of non-mammalian hosts such as the fly.

Ingestion of V. cholerae Results in Lethal Infection of D. melanogaster
To test the utility of D. melanogaster as a model host for V. cholerae, flies were fed either Luria-Bertani (LB) broth alone or inoculated with V. cholerae. Consumption of this growth medium by the fly was documented on multiple occasions by addition of blue dye. Using this experimental design, wildtype flies fed LB broth alone survived for 5 d and could be maintained for up to 2 wk if a larger volume of LB broth was provided. In contrast, flies fed LB inoculated with V. cholerae expired after 3 d regardless of the amount of volume provided ( Figure 1). Similar observations were made for the Canton-S wild-type strain of D. melanogaster and for several D. melanogaster strains carrying benign marker mutations (unpublished data).

V. cholerae Is Able to Multiply within the Fly
Once ingested by a model mammalian host, V. cholerae is able to multiply within the intestinal compartment [34]. In the experimental model presented above, flies were contin-uously fed V. cholerae. While this type of infection is rapidly lethal, it does not distinguish between bacterial accumulation and bacterial colonization and multiplication. To test whether V. cholerae was able to persist and multiply within the fly, we measured V. cholerae colony-forming unit (CFU)/fly over time in flies continuously fed LB inoculated with V. cholerae and in flies first fed LB inoculated with V. cholerae for 24 h and then transferred to a vial containing sterile LB broth. At 24 h, flies in both groups harbored equivalent numbers of V. cholerae. As shown in Figure 2A, flies exposed continuously to LB inoculated with V. cholerae expired after 3 d when the burden of V. cholerae reached 3.93 3 10 7 CFU/fly. Over the course of 4 d, numbers of V. cholerae also increased in flies removed from contaminated food, albeit at a slower rate than flies continuously exposed to V. cholerae. The number of V. cholerae required to bring about death was similar in both groups. These results suggest that V. cholerae is able to colonize and multiply within the fly in the absence of continued ingestion.

V. cholerae Remains Localized to the Fly Gut following Ingestion
During human infection, V. cholerae remains localized to the intestine, causing systemic disease through the action of cholera toxin. To determine whether V. cholerae also remained localized to the Drosophila gut, whole flies fed either sterile LB or the V. cholerae/LB mixture were processed into 5-lm thick histologic sections, stained, and examined. Many slender, comma-shaped, gram-negative rods were found within the midgut of V. cholerae-infected flies ( Figure 2B). Although concentrated in the midgut, V. cholerae were also found in other regions of the gut. Careful histologic analysis of all tissues revealed no V. cholerae outside the fly alimentary tract. Interestingly, the intestines of flies fed both sterile LB, and LB inoculated with V. cholerae contained gram-positive rods

Synopsis
Cholera, the pandemic diarrheal disease caused by the gramnegative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods, and insects have previously been implicated as vectors of this disease. Here researchers report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease. Furthermore, although ingestion of cholera toxin results in massive diarrhea in mammals, these researchers have found that ingestion of purified cholera toxin is not lethal to the fly. However, when co-ingested with a pathogenic strain of V. cholerae carrying a deletion of the cholera toxin genes, cholera toxin is lethal. These findings not only demonstrate the utility of D. melanogaster as an accurate, inexpensive model for elucidation of the host-pathogen interaction and identification of inhibitors of the action of cholera toxin; they also suggest that V. cholerae carries additional virulence factors that enable intoxication of an arthropod host. Based on these findings, the researchers suggest that the fly or a related arthropod may be a true host of V. cholerae in nature.
( Figure 2C). These most likely represent the commensal flora of our laboratory flies.

Cholera Toxin Is a Virulence Factor in V. cholerae Infection of the Fly
We hypothesized that, as is the case in human disease, cholera toxin secreted from V. cholerae within the fly gut was responsible for death. To test this hypothesis, a V. cholerae mutant harboring a deletion in the ctxB gene was constructed and fed to wild-type flies [35]. The DctxB mutant was significantly less virulent in the fly model of cholera, demonstrating that cholera toxin is the primary virulence factor in V. cholerae infection of both flies and humans ( Figure  1). Although flies fed a DctxB mutant survived several days longer than flies fed wild-type V. cholerae, they still died prematurely. Thus, we hypothesize that, in the absence of cholera toxin, other virulence factors contribute to death of the fly.

V. cholerae-Infected Flies Lose Weight Prior to Death
Cholera victims may lose 10% or more of their body weight due to dehydration as a result of secretory diarrhea [36]. If cholera toxin acts via a similar mechanism in the fly, weight loss should also occur during infection of the fly. To test this, flies fed either LB alone or LB inoculated with V. cholerae were weighed on a daily basis. Over the course of 3 d, flies fed V. cholerae lost approximately 25 % of their initial body weight, while flies fed LB alone showed a small weight gain ( Figure 3). These results support the hypothesis that flies, like humans, become dehydrated during V. cholerae infection. However, we cannot exclude other causes of weight loss such as a decreased food intake or altered metabolic activity.  Cell culture-based studies have shown that G sa , adenylyl cyclase, and the KCNN4 channel play an important role in V. cholerae-induced Cl À secretion by intestinal epithelial cells [9,37,38]. We asked whether these same factors might be required for susceptibility of Drosophila to V. cholerae infection by examining the susceptibility of Drosophila strains bearing mutations in the genes encoding G-sa60A, the adenylyl cyclase rutabaga, or the SK channel, a Ca 2þ -sensitive K þ channel that is the closest Drosophila homolog of the human KCNN4 channel. As shown in Figures 4 and 5A, mutation of G-sa60A and rutabaga provided nearly complete protection against V. cholerae infection. Mutation of Sk provided only partial protection. This may be the result of persistent, albeit reduced expression of the SK channel in this mutant or of additional mechanisms that facilitate Cl À secretion in the fly ( Figure 6). Importantly, we confirmed that the additional independently generated mutant alleles for G-sa60A, rut, or SK listed in Table 1 had similar effects on V. cholerae susceptibility, indicating that mutations in these genes, rather than other differences in genetic background, caused the observed phenotypes.
In preparation for genetic rescue of the rut mutant phenotype using the GAL4/UAS binary expression system, a rut 2080 strain homozygous for a UAS-rut þ transgene insertion on the second chromosome was obtained and assayed for susceptibility to V. cholerae infection [39]. Unexpectedly, these flies were susceptible ( Figure 5A). To ascertain the basis of this susceptibility, we assayed levels of rut transcript in wildtype, rut 2080 , and rut 2080 ;UAS-rut þ flies by RT-PCR. As shown in Figure 5B, rut transcription was greatly reduced in the rut 2080 mutant, but the rut 2080 ;UAS-rut þ flies had transcript levels comparable to those of wild-type flies. PCR analysis con-  firmed the presence of the rut 2080 insertion in both strains. Thus, we conclude that the UAS-rut transgene is transcribed in the absence of Gal4, presumably by regulation from an adjacent genomic element. Furthermore, we conclude that susceptibility of rut mutant flies to V. cholerae infection is rescued by restoration of wild-type levels of the rutabaga transcript.

Clotrimazole Protects V. cholerae-Infected Flies against Death
Because clotrimazole abrogates the V. cholerae-induced secretory diarrhea in mammals by inhibiting K þ transport through KCNN4 channels, we postulated that co-administration of clotrimazole with V. cholerae might also block K þ transport through the Drosophila SK channel and, therefore, protect wild-type flies against death. Figure 6 shows that this was indeed the case. However, co-administration of clotrimazole had no effect on survival of SK mutant flies, suggesting that clotrimazole is, in fact, exerting its effect by interaction with the SK channel ( Figure 6).

A Factor Carried by Pathogenic V. cholerae Is Required for Intoxication of the Fly by Cholera Toxin
Ingestion of cholera toxin is sufficient to cause massive intestinal fluid accumulation and diarrhea in mammals [30][31][32][33]. Thus, we predicted that ingestion of purified, active cholera toxin alone would result in death of the fly. Remarkably, ingestion of LB containing as much as 100 lg/ ml of cholera toxin did not alter survival of the fly (unpublished data). We questioned whether the presence of V. cholerae itself might be required for intoxication of the fly by cholera toxin. To test this, we fed LB containing both cholera toxin and a V. cholerae DctxB mutant to flies. As shown in Figure 7, ingestion of cholera toxin in the presence of the DctxB mutant V. cholerae resulted in death of the flies at rates similar to those of flies infected with wild-type V. cholerae alone. This suggested to us that an unknown bacterial factor might be required for intoxication of the fly by cholera toxin. To determine whether this factor might be specific to pathogenic isolates of V. cholerae, we fed LB containing cholera toxin and one of several non-toxigenic environmental isolates of V. cholerae to flies. In each case, there was no significant difference in survival between flies fed V. cholerae alone and those fed V. cholerae combined with cholera toxin. To test whether this cholera toxin-potentiating factor was carried on the CTXU, we combined cholera toxin with Bengal2, a pathogenic strain of V. cholerae carrying a deletion of the CTXU. This mutant was also able to provide the flyspecific virulence factor (unpublished data). Thus, this factor is not carried on the CTXU. These experiments suggest that pathogenic V. cholerae possess a virulence factor or factors that are essential for intoxication of arthropods but not mammals by cholera toxin.

Implications of this Model for the Study, Treatment, and Ecology of Cholera
We have demonstrated surprising parallels in the mechanism of V. cholerae-mediated death of man and the model arthropod D. melanogaster. Cholera toxin is the primary virulence factor in both infections. While the mechanism of cholera toxin has previously been elucidated in cultured intestinal epithelial cells, we present the first evidence that this mechanism is also operative in whole organisms. Furthermore, this model system will have wide-ranging applications to the study of this devastating disease. Due to the expense and labor involved in mammalian genetic screens, little is known about the host factors that govern susceptibility to cholera. Because lethal oral infection of the fly requires no manipulation by the experimentalist and has an easily measured outcome, the fly provides a powerful tool  to be used in large-scale genetic screens for host susceptibility factors and bacterial virulence factors. The current mainstay of cholera therapy consists of administration of oral or intravenous water and ions until the infection is overcome by antibiotics and /or the innate immune system. An inhibitor of the secretory diarrhea caused by cholera toxin would be a potentially life-saving adjuvant to this therapy. We have shown here that oral agents can block the action of cholera toxin in the fly. Thus, this model will also facilitate screens of combinatorial chemical libraries for inhibitors of cholera toxin and secretory diarrhea. Finally, these studies highlight a host-pathogen interaction that could easily occur in nature. Close contact between V. cholerae and arthropods has been documented and is likely more frequent than that between V. cholerae and humans [19,[40][41][42]. In fact, environmental studies have demonstrated that common house flies carry V. cholerae in endemic areas [22][23][24][25]. In this work, we have presented evidence that pathogenic V. cholerae carry virulence factors that are essential for intoxication of the fly but not mammals. Thus, we present the provocative hypothesis that the pathogenic program of V. cholerae may have evolved for an arthropod rather than for us.

Materials and Methods
Bacterial strains, fly strains, and growth media. MO10, a V. cholerae O139 clinical isolate, and mutants derived from this strain were used in all experiments [43]. All fly strains were reared at room temperature on standard Drosophila media. The wild-type OregonR fly strain was used for most studies. Gsa, rut, and Sk experiments utilized mutant fly lines harboring G-sa 60A R60 , a loss-of-function allele that reduces the cAMP concentration 4-to 5-fold in larvae [44], rut 2080 , an enhancer trap element in the 5 9 flanking region of the rut gene [45], and PBacfWHgSK f07979 , respectively ( Table 1). The rut 2080 and rut 2080 ;UAS-rut þ fly lines were generously provided by Ron Davis. The presence of the rut 2080 mutant allele was confirmed by PCR amplification of a portion of the insertion element for both lines. Additionally, fly lines carrying G-sa60A B19 , PfEPgrut EP399 or PfGT1grut BG00139 , and PfSUPor-PgKG00471 or PfGT1gSK BG01378 were used to confirm the results of experiments with the G-sa60A R60 , rut 2080 , and PBacfWHgSK f07979 mutant fly strains, respectively (Table  1). Fly lines other than those noted were obtained from the Bloomington Drosophila Stock Center (Bloomington, Indiana).
V. cholerae mutant construction. The V. cholerae DctxB mutant, harboring a 321 bp deletion in the ctxB gene (VC1456) was constructed by double homologous recombination according to previously described protocols [35]. The deletion removed all but 11 amino acids remaining at the amino-terminus of the protein and the terminal stop codon.
Survival of Drosophila following V. cholerae infection. Ten wild-type Oregon-R adult flies were placed in each of three vials containing a cotton plug saturated with Luria-Bertani (LB) broth either alone or inoculated with 10 8 CFU/ml of V. cholerae O139 strain MO10 or another strain as noted in the text [46]. Viable flies were counted at 24-h intervals. Reproducibility of all survival curves was confirmed in at least three independent experiments, and log-rank tests were used to determine statistical significance.
Histological studies. Flies were fed either LB inoculated with V. cholerae or LB alone for 48 h, and then anesthetized and fixed in formalin for 48 h prior to processing. Flies were processed on a tissue processor (Leica ASP 300, Wetzlar, Germany) and embedded in paraffin. The embedded flies were sectioned into 5-lm ribbons, which were placed on positively charged glass slides, baked at 65 8C overnight, and gram stained.
Weight loss measurements. Sets of ten female flies were weighed and then transferred to fly vials containing either LB alone or LB inoculated with V. cholerae. Flies, housed in thin-walled Eppendorf tubes, were weighed 24 and 48 h after transfer, using a precision balance (Mettler Toledo AG204, Columbus, Ohio). All experiments were performed in triplicate, and the average ratios of final to initial weight were calculated.
Quantification of V. cholerae within flies. To determine whether V. cholerae was able to colonize and multiply within the fly, flies fed either LB alone or LB inoculated with V. cholerae were anesthetized, removed from vials, and homogenized in LB broth at 24-h intervals. Particulates were pelleted, and dilutions of the resulting supernatants were plated on LB-agar supplemented with streptomycin (100 mg/ml). In all cases, no colonies were obtained from LB-fed flies.
Fly intoxication with purified cholera toxin. Adult Oregon-R flies 3-5 d old were fed cholera toxin diluted to the specified concentrations in LB broth. Overnight cultures containing V. cholerae strains were also added to the mixture in a 1:10 dilution where specified. Flies were monitored at 24-h time intervals until death. Survival of flies was plotted against time using Kaplan-Meier plots, and a logrank test was performed to determine statistical significance.