RESUMO
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, Gsalpha, 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 mug 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.
RESUMO
Dopamine is an important neurotransmitter in the central nervous system of both Drosophila and mammals. Despite the evolutionary distance, functional parallels exist between the fly and mammalian dopaminergic systems, with both playing roles in modulating locomotor activity, sexual function, and the response to drugs of abuse. In mammals, dopamine exerts its effects through either dopamine 1-like (D1-like) or D2-like G protein-coupled receptors. Although pharmacologic data suggest the presence of both receptor subtypes in insects, only cDNAs encoding D1-like proteins have been isolated previously. Here we report the cloning and characterization of a newly discovered Drosophila dopamine receptor. Sequence analysis reveals that this putative protein shares highest homology with known mammalian dopamine 2-like receptors. Eight isoforms of the Drosophila D2-like receptor (DD2R) transcript have been identified, each the result of alternative splicing. The encoded heptahelical receptors range in size from 461 to 606 aa, with variability in the length and sequence of the third intracellular loop. Pharmacologic assessment of three DD2R isoforms, DD2R-606, DD2R-506, and DD2R-461, revealed that among the endogenous biogenic amines, dopamine is most potent at each receptor. As established for mammalian D2-like receptors, stimulation of the Drosophila homologs with dopamine triggers pertussis toxin-sensitive Gi/o-mediated signaling. The D2-like receptor agonist, bromocriptine, has nanomolar potency at DD2R-606, -506, and -461, whereas multiple D2-like receptor antagonists (as established with mammalian receptors) have markedly reduced if any affinity when assessed at the fly receptor isoforms. The isolation of cDNAs encoding Drosophila D2-like receptors extends the range of apparent parallels between the dopaminergic system in flies and mammals. Pharmacologic and genetic manipulation of the DD2Rs will provide the opportunity to better define the physiologic role of these proteins in vivo and further explore the utility of invertebrates as a model system for understanding dopaminergic function in higher organisms.
