Research Interest: Host-Pathogen Interactions, In vivo models of intoxication, Cellular microbiology

Disease-causing bacteria produce virulence factors which allow the pathogen to gain an advantage overall the host during illness.Thus, the study and identification of virulence factors provides important insights, and reveals therapeutic targets, which advance our understanding of mechanisms of pathogenesis.Experiments in our laboratory focus on characterizing the mechanism of action for virulence factors produced by Bacillus anthracis and Clostridium difficile.

Danio rerio as a model for identifying the systemic impact of intracellular bacterial toxins.

The mechanism of action for several bacterial toxins has been characterized at the atomic, molecular and cellular levels.Unfortunately, in most cases it has been difficult to determine how these activities apply to changes in the overall physiology of the infected individual, since the organs targeted by these proteins are not known.For this reason, we have taken advantage of developing zebrafish embryos in order to develop leads on organs targeted by these toxins.Zebrafish embryos are translucent and internal organs can be directly visualized by standard light microscopy.Recently, we have used this model to show that B. anthracis edema toxin, a calmodulin-dependent adenylate cyclase, causes severe tissue necrosis, which most prominent in the gastrointestinal tract.

In addition, we have used a similar approach to provide insight into the major organs impacted by Clostridium difficile toxin B.C. difficile causes a life-threatening illness, and is the leading cause of hospital-acquired diarrhea, and illness occurs by the production of two potent exotoxins.Toxin B is responsible for the systemic, and life-threatening aspects of this illness, yet the major organs targeted by this toxin are not known.Using the zebrafish embryo model we have discovered that toxin B is a potent cardiotoxin, and blocks appropriate ventricle function and reduces blood flow.Studies are ongoing to determine the domains of this protein responsible for localizing this toxin to the heart, and to identify the particular substructures of the heart impacted by this important virulence factor.

Characterization of signaling pathways impacted by TcdB and their involvement in cell death.

In order to cause cell death, toxin B functions as an intracellular bacterial toxin and inactivates small GTPases (Rho, Rac and Cdc42) within the cell.To date, little is known about the specific mechanism of cell death, which occur following inactivation of the small GTPases.Rho, Rac and Cdc42 regulate cell structure and cell survival.In recent studies experiments have focused on elucidating the disrupted signaling events leading to death of TcdB intoxicated cells.We are currently using a combination of cell-based experimental approaches to better define this event.Our longer term goal is to combine this information with findings from the zebrafish study in order to provide a comprehensive picture of TcdB cellular and systemic effects.

B. anthracis host interactions

During early stages of anthrax disease, B. anthracis spores are engaged and engulfed by resident macrophages, which subsequently traffic spores to regional lymph nodes.Thus, dissecting B. anthracis spore-macrophage interactions is critical to understanding early events in anthrax disease.Our work has focused on macrophage related aspects of this interaction.Among the primary candidates for regulating this interaction on the part of the macrophage are MAPK signaling pathways.In a series of experiments we have recently uncovered the role of p38 signaling in this interaction and have shown that this signaling pathway is necessary for reducing the survival of B. anthracis within the cell.Experiments are now focused on identifying the important the cellular factors regulated by p38 duringmacrophage interaction with B. anthracis.Using a combination of proteomic and DNA microarray approaches, we are identifying important transcriptional and translational changes regulated by p38 and are determining how these events contribute to reducing the growth of B. anthracis within the cell.

Recent Publications:

Salles, I.I., Tucker, Amy. E., Voth D.E. and J.D. Ballard (2003) Toxin-induced resistance in Bacillus anthracis lethal toxin-treated macrophages. Proceedings of The National Academy of Sciences100(21) 12426-12431

Mourez, M., Yan, M., Lacy, D.B., Dillon, L., Betnsen, L., Marpoe, A., Maurin, C., Hotze, E., Wiggleworth, D., Pimetal, R-A, Ballard, J.D., Collier, R.J. and R. K. Tweten (2003) Mapping Dominant-negative mutations of anthrax protective antigen by scanning mutagenesis. Proceedings of The National Academy of Sciences, 100 (24) 13803-13808

Voth, D.E., Hamm. E, and J.D. Ballard (2004) Clostrdium sordellii lethal toxin is maintained in a multimeric protein complex. Infection and Immunity 72 (6):3366-72

Tucker, A and J.D. Ballard (2005) Anthrax toxin and genetic aspects regulating its expression.Topics in Current Genetics, 11, 21-34

Voth, D., and J.D. Ballard (2005) Clostridium difficile toxins: Mechanism of Action and Role in Disease, Clinical Microbiological Reviews, 18 (2), 247-263

Voth, D.E., Hamme, E.E., Nguyen,L.G.,Tucker, A.E., Salles, I.I., Ortiz-Leduc, W., and J.D. Ballard (2005) Bacillus anthracis Edema Toxin as a Cause of Tissue Necrosis and Cell-Type Specific Cytotoxicity. Cellular Microbiology, 7 (8), 1139-1149

Salles, I.I., Voth, D.E. Ward, S.C., Averette, K.M., Tweten, R.K., Bradley, K.A., and J.D. Ballard (2006)Cytotoxic Activity of Bacillus anthracis Protective Antigen Observed in a Macrophage Cell Line Overexpressing ANTXR1. Cellular Microbiology, (8), 1272-1281

E. E. Hamm, D. E. Voth, and J.D. Ballard (2006) Identification of Clostridium difficile TcdB cardiotoxicity using a zebrafish embryo model of intoxication. Proceedings of The National Academy of Sciences U S A 103, (38), 14176-81.

Hamm, E.E. and J. D. Ballard (2007) Elucidating the in vivo targets of bacterial toxins, Future Microbiology 2(1) 85-92

Dodd, D., Reese, J.G., Louer, C.R., Ballard, J.D., Spies, M.A., and S.R. Blanke (2007) Functional comparison of the two Bacillus anthracis glutamate racemases, J. Bacteriol, 189(14):5265-75

Voth, D.E. and J.D. Ballard (2007) Critical intermediate steps in Clostridium sordellii lethal toxin-induced apoptosis.Biochem. Biophys. Res. Commun. in press

McKevitt, M.T., Bryant, K.M., Shakir, S., Larabee, J.L., Blanke, S.R., Lovchik, J., Lyons, C.R., and J.D. Ballard (2007) Endogenous D-alanine Synthesis and Auto-Inhibition of Bacillus anthracis Germination: Effects on in vitro and in vivo infections. in press

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