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.
Presently my laboratory is studying the structure and function relationships
of the cytolethal distending toxin (CDT) of E. coli. CDT gets its name from
the fact that that toxin treatment of cultured cells results in massive cellular 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.
 |
Model for CDT Action
(click to enlarge) |
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.
 |
| (click to enlarge) |
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.
