Our recent discovery that key virulence factors from Bacillus anthracis, edema factor (EF), and Vibrio cholerae, cholera toxin (Ctx), both inhibit protein trafficking to cell-cell junctions is paradigm shifting. EF is a highly active adenylate cyclase and Ctx ADP-ribosylates Gs? subunits to constitutively activate host adenylate cyclase. These two cAMP producing toxins reduce the levels and activity of a small GTPase (Rab11) required in the final step of endocytic recycling of cell adhesion molecules (e.g., cadherins) and signaling proteins (e.g., Notch components) to cell-cell junctions, resulting in disruption of the vascular endothelium (EF) or intestinal epithelium (Ctx). This novel effect of EF and Ctx was discovered and genetically dissected in the model system Drosophila melanogaster (fruit fly), and these cell biological mechanisms translate to toxin action in human vascular endothelial cells (EF) and intestinal epithelial cells (Ctx) as well as in vivo in mice (both EF and Ctx). Another key finding, with important practical implications, was that over-expression of Rab11 can reverse the junction disrupting effects of EF and Ctx in vivo in flies and in human cells. In the current revised grant, we propose three integrated aims to elucidate the pathways mediating the barrier disruptive actions of EF and Ctx and to analyze the consequences of this novel cell biological mechanism in disease pathogenesis.
In Aim 1 we will examine the pathways by which high sustained levels of cAMP produced by EF/Ctx reduce Rab11 protein levels to derail junctional transport and explore new potential functions of EF and Ctx related to inhibition of exocyst function in immune cells.
In Aim 2, we will investigate how inhibition of endocytic recycling promotes leakage across human cell monolayers and in the vasculature during anthrax infection. Since vascular collapse is a frequent cause of death in anthrax, we will also determine whether increasing Rab11 levels or treating with known traffic-promoting agents can reverse the vascular leakage caused by EF.
In Aim 3, we will similarly examine the contribution of exocyst inhibition to the massive fluid secretion that is pathognomonic of cholera and whether elevating endocytic recycling via genetic or pharmacological means is protective in vivo. The proposed studies have important translational relevance to treating anthrax since toxins can reach critical lethal levels just as patients begin to seek medical intervention, when antibiotics are no longer effective. Thus, treatments based on restoring endocytic recycling could be used in conjunction with existing anti-toxin therapies (e.g., anti-toxin antibodies, small molecule inhibitors) to neutralize toxins already present in the circulation. An advantage of traffic-promoting agents is that they would intervene at the very last step when vascular integrity collapses and other organ systems fail. Such traffic-promoting compounds might also increase the efficacy of fluid replacements to treat cholera and to treat other barrier disruptive diseases including: ischemia, asthma, dermatitis, IBD, cancer, ciliary diseases, and neurodegenerative disorders.
Bacillus anthracis (B.a.), a foremost bioterrorism threat, and Vibrio cholera (V.c.), a global pandemic pathogen, each produce a toxin essential to disease pathogenesis that markedly elevates cAMP levels in host cells: B.a. edema factor (EF) is a highly active adenylate cyclase, and V.c. cholera toxin (Ctx) ADP-ribosylates host Gsa proteins to constitutively activate host adenylate cyclase. We recently discovered that these two toxins inhibit endocytic recycling to cell-cell junctions, disrupting barrier integrity of the vascular endothelium (EF) and intestinal epithelium (Ctx), respectively. In the current grant, we will dissect the genetic pathways by which these cAMP toxins exert their barrier disruptive effects and test whether elevating endocytic recycling by genetic means or pharmacologically is protective, the latter objective having broad translational relevance to many human diseases in which barrier function is disrupted by infection of inflammatory processes.
|Gantz, Valentino M; Jasinskiene, Nijole; Tatarenkova, Olga et al. (2015) Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc Natl Acad Sci U S A 112:E6736-43|
|Gantz, Valentino M; Bier, Ethan (2015) Genome editing. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations. Science 348:442-4|
|Akbari, Omar S; Bellen, Hugo J; Bier, Ethan et al. (2015) BIOSAFETY. Safeguarding gene drive experiments in the laboratory. Science 349:927-9|
|Gonzalez, David J; Corriden, Ross; Akong-Moore, Kathryn et al. (2014) N-terminal ArgD peptides from the classical Staphylococcus aureus Agr system have cytotoxic and proinflammatory activities. Chem Biol 21:1457-62|
|Guichard, Annabel; Nizet, Victor; Bier, Ethan (2014) RAB11-mediated trafficking in host-pathogen interactions. Nat Rev Microbiol 12:624-34|