The overall focus of my research is to define a better understanding how bacterial toxins function as virulence factors. Bacterial toxins are macromolecules produced by numerous disease-causing microorganisms that often exert lethal or dramatic effects on eukaryotic cells. In some cases, bacterial toxins are solely responsible for the pathological effects of a microbe on its host; many toxins however affect the host in more subtle ways that contribute to the overall disease state. By exploring the structure and function relationships of bacterial toxin, we hope to better understand how they exert their biological effects on human cells and in effect, ascertain their role in disease. In addition, the study of bacterial toxins has often provided valuable insights into mammalian cell biology.
My laboratory studies the structure and function relationships of the cytolethal distending toxin (CDT) of E. coli. CDT gets its name from the fact that toxin treatment of cultured cells results in cellular distension or swelling, actin reassembly in some mammalian cell types, and eventual cell death. CDT is composed of three proteins, CdtA, CdtB and CdtC, all three of which are required for biological activity. The hallmark of CDT action on cells is an irreversible block in the cell cycle at the G2/M boundary resulting from inactivation, by persistent phosphorylation, of the cell cycle regulatory kinase cdc2. Inactivation of cdc2 prevents entry into mitosis thus CDT-treated cells display a characteristic block at G2. Cell death appears to be a function of serious chromosomal abnormalities, apoptosis and/or endoreduplication. The effects of CDT on cells resembles that of ionizing radiation which led to the discovery that CdtB, the biologically active CDT subunit is a homolog of mammalian type I DNase.
Previous studies from my laboratory discovered the nuclease activity associated with CdtB and the relationship of enzymatic activity to biological function. We also characterized the nuclear translocation of E. coli CdtB and the dependence of translocation on bipartite nuclear localizing signals found in the CdtB primary sequence. Mutational analysis and functional studies on the CdtB NLS sequences indicate that, as expected, nuclear import is absolutely required for cellular intoxication.
All three subunits of CdtB are required for biological activity. The CdtA and CdtC subunits are lectins that bind carbohydrate structures on the target cell surface. Both subunits appear to bind the same carbohydrate structure, but the CdTAC dimer is absolutely required for proper binding to the CDT receptor. Binding of CdtAC mediates subsequent internalization of CDT thus allowing CdtB access to the nuclear envelope. Presently, our studies on CdtA and CdtC are directed towards determining the cell surface receptor for the holotoxin.
We are also investigating the molecular structure and dynamics of CdtB in hopes of better characterizing nuclear translocation and the interaction of CdtB with its biologically relevant substrate, chromosomal DNA.
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