Campylobacter jejuni is a food and water borne pathogen responsible for severe inflammatory diarrhea in humans. C. jejuni is a NIAID Category B Priority Pathogen because, in part, of the potential of this pathogen to be used as a biowarfare agent to contaminate food- and water supplies over large geographical areas. It is important to understand the basic biology of C. jejuni pathogenesis, which may lead to novel strategies for circumventing the use of this bacterium as a biowarfare agent. However, the pathogenic mechanisms used by C. jejuni to colonize and cause disease in the host are poorly understood. This application is a collaborative proposal between investigators at the University of Houston and UCLA to study the only known exotoxin secreted by C. jejuni. We will investigate the cellular mechanisms of the C. jejuni cytolethal distending toxin (CDT), which causes cell cycle arrest and eventual death of intoxicated mammalian cells, and has been proposed to assist in remodeling the in vivo environment to facilitate colonization of the intestinal tract. In this R21 application, we will begin to explore the hypothesis that CDT discriminates for and binds to a specific plasma membrane receptor on the surface of sensitive cells as an essential early step during cellular intoxication. In this application, we propose experiments for investigating at the molecular level the interactions of CDT with target cells.
In Specific Aim 1, University of Houston researchers will characterize the interactions of CDT with sensitive mammalian cell lines. We will determine the specific and non-specific components as well as the affinity of CDT-receptor interactions. In addition, we will establish many how receptors are present per mammalian cell. Moreover, because CDT is a tri-partite toxin comprising three discrete subunits (CdtA, CdtB, and CdtC), we will establish the contribution of each subunit to binding of the toxin. Finally, we will begin to characterize the nature of the CDT receptor. These experiments will be important for establishing the framework for future experiments to identify the molecular basis for CDT receptor discrimination and binding.
In Specific Aim 2, we will identify and characterize mutant cell lines that are resistant to CDT. UCLA researchers will use a genetic approach involving two fundamental phases. In phase 1, cell lines will be mutagenized and screened for a loss of sensitivity to CDT. In phase 2, the loss of sensitivity will be characterized, with the goal being the identification of a cell line that is deficient in binding to CDT. These experiments will be crucial for future work to complement the cell-binding defect, which will identify the putative CDT receptor. Results from this research will provide important information about the mechanism of CDT cellular intoxication, and will provide the basis for future work to develop strategies for blocking the action of CDT as an approach for attenuating C. jejuni pathogenesis.
| Gargi, Amandeep; Tamilselvam, Batcha; Powers, Brendan et al. (2013) Cellular interactions of the cytolethal distending toxins from Escherichia coli and Haemophilus ducreyi. J Biol Chem 288:7492-505 |
| Eshraghi, Aria; Maldonado-Arocho, Francisco J; Gargi, Amandeep et al. (2010) Cytolethal distending toxin family members are differentially affected by alterations in host glycans and membrane cholesterol. J Biol Chem 285:18199-207 |
| Banks, David J; Bradley, Kenneth A (2007) SILENCE: a new forward genetic technology. Nat Methods 4:51-3 |
| Maldonado-Arocho, Francisco J; Fulcher, Jennifer A; Lee, Benhur et al. (2006) Anthrax oedema toxin induces anthrax toxin receptor expression in monocyte-derived cells. Mol Microbiol 61:324-37 |
| Banks, David J; Barnajian, Moshe; Maldonado-Arocho, Francisco J et al. (2005) Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge. Cell Microbiol 7:1173-85 |
