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

Identification of a Human Monoclonal Antibody to Replace Equine Diphtheria Anti-toxin for the Treatment of Diphtheria

Diphtheria anti-toxin (DAT) has been used to treat Corynebacterium diphtheriae infection for over one hundred years. While the global incidence of diphtheria has declined in the 20th century, the disease remains endemic in many parts of the world and significant outbreaks still occur. Diphtheria anti-toxin is an equine polyclonal antibody with considerable side effects that is in critically short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we cloned human monoclonal antibodies (HuMabs) directly from antibody secreting cells of human volunteers immunized with Td vaccine. We isolated a diverse panel of HuMabs that recognized diphtheria toxoid and recombinant protein fragments of diphtheria toxin. Forty-one unique HuMabs were expressed in 293T cells and tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays. The lead candidate HuMab, 315C4 potently neutralized diphtheria toxin with an EC50 of 0.65 ng/mL. Additionally, 25 μg of 315C4 completely protected guinea pigs in an in vivo lethality model. In comparison, 1.6 IU of DAT was necessary for full protection resulting in an estimated relative potency of 64 IU/mg for 315C4. We further established that our lead candidate HuMab binds to the receptor binding domain of diphtheria toxin and blocks the toxin from binding to the putative receptor, heparin binding-epidermal growth factor like growth factor. The discovery of a specific and potent neutralizing antibody against diphtheria toxin holds promise as a potential human therapeutic and is being developed for human use

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

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

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

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

Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus▿

Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation