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

L'organisation ribonucleoproteieque des snRNAS de la cellule HeLa

SIGLECNRS T 57433 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Ingestion of <i>V. cholerae</i> Induces <i>Drosophila</i> Weight Loss

<p>Fraction of initial weight gained by wild-type flies (wt Dm) fed either LB alone (LB) or <i>V. cholerae</i> (wt Vc). Error bars represent the standard deviation based on three measurements.</p

The Genes Encoding Cholera Toxin Are Required for Lethal <i>V. cholerae</i> Infection of <i>Drosophila</i>

<p>Fractional survival of wild-type Oregon R flies (wtDm) fed LB alone (LB), wild-type <i>V. cholerae</i> (wtVc), or a <i>V. cholerae</i> Δ<i>ctxB</i> mutant (ctxB). Ten adult flies (five males and five females), 3–5 d following eclosion were used. Log-rank test analysis demonstrated a statistically significance difference in survival of wild-type <i>V. cholerae</i> infected flies and <i>V. cholerae</i> Δ<i>ctxB</i> mutant infected flies (<i>p</i> < 0.0001).</p

A Bacterial Factor Is Required for Intoxication of the Fly by Cholera Toxin

<p>Fractional survival over time of wild-type flies fed LB alone, wild-type <i>V. cholerae,</i> or a <i>V. cholerae</i>Δ<i>ctxB</i> mutant (ctxB) either with or without 10 μg/ml purified cholera toxin. Log-rank test analysis demonstrated a statistically significant difference in the survival of wild-type flies fed a <i>V. cholerae</i> Δ<i>ctxB</i> mutant (ctxB) alone and those fed a <i>V. cholerae</i> Δ<i>ctxB</i> mutant (ctxB) combined with purified cholera toxin (<i>p</i> < 0.0001).</p

<i>V. cholerae</i> Multiplies within the Gut of the Fly following Infection

<div><p>(A) Colony counts were assayed at 24-h time points from flies infected with <i>V. cholerae.</i> Grey bars indicate CFU per fly obtained from flies fed <i>V. cholerae</i> continuously, while black bars depict CFU per fly for flies fed <i>V. cholerae</i> for 24 h and then removed to a sterile, fresh LB solution.</p><p>(B) Section of the midgut of a fly harvested 48 h after introduction to medium containing <i>V. cholerae</i>. Arrows labeled with Vc point to clusters of slender, curved gram negative <i>V. cholerae</i> (pink) present in the lumen of the midgut of the infected fly. Occasional gram positive bacteria (violet), which represent the endogenous flora of the gut, are also present.</p><p>(C) Section of the midgut of a fly harvested 48 h after introduction to LB alone. Only endogenous gram positive bacteria (violet) could be observed in the intestines of flies fed sterile LB broth.</p></div

SK Mutant <i>Drosophila a</i>nd Clotrimazole-Treated Wild-Type Flies Display Partial Resistance to Lethal <i>V. cholerae</i> Infection

<p>Fractional survival over time of wild-type (wt Dm) or <i>SK</i> mutant ({WH}SK^f07979) flies fed either wild-type <i>V. cholerae</i> alone or combined with 10 μg/ml clotrimazole (10 μg Clot). Log-rank test analysis demonstrated a statistically significant difference in survival of wild-type flies fed wild-type <i>V. cholerae</i> and <i>SK</i> mutant ({WH}SK^f07979) flies fed wild-type <i>V. cholerae</i> (<i>p</i> < 0.0001). There was also a statistically significant difference in survival of wild-type flies fed wild-type <i>V. cholerae</i> and wild-type flies fed wild-type <i>V. cholerae</i> combined with 10 μg/ml clotrimazole (<i>p</i> < 0.0001).</p

A <i>rut²⁰⁸⁰</i> Mutant Strain Is Resistant to Lethal <i>V. cholerae</i> Infection

<div><p>(A) Fractional survival over time of wild type flies, <i>rut²⁰⁸⁰</i> mutant flies [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010008#ppat-0010008-b47" target="_blank">47</a>], and <i>rut²⁰⁸⁰</i><i>UAS-rut⁺</i> fed LB inoculated with <i>V. cholerae</i> (wt Vc). Wild-type flies fed LB broth alone were included as a control. Log-rank test analysis demonstrated a statistically significant difference in the survival of wild-type flies fed wild-type <i>V. cholerae</i> and <i>rut²⁰⁸⁰</i> mutant flies fed wild-type <i>V. cholerae</i> (<i>p</i> < 0.0001).</p><p>(B) RT-PCR amplification of <i>rutabaga</i> transcripts in wild-type (WT), <i>rut²⁰⁸⁰</i>, and <i>rut²⁰⁸⁰ UAS-rut</i>⁺ flies. The ribosomal protein <i>rp15a</i> was used as a constitutively transcribed control.</p></div