Actinobacillus actinomycetemcomitans(Aa), a major pathogen in certain human periodontal diseases, elaborates a potent leukotoxin (Ltx). This polypeptide kills human neutrophils, monocytes and T lymphocytes and may impair host antibacterial resistance by destroying or perturbing these cells in infected tissues. Results of current studies indicate that the toxin disrupts the permeability barrier of the plasma membrane of susceptible cells. This results in a 2+rapid rise in Ca (and probably other ions) levels within the cell which ultimately leads to cell death. This proposal will determine the molecular mechanism(s) for this observation. Two separate lines of experimentation form the basis for this proposal: l) Ltx-treated lymphocytes and NK cells exhibit many of the morphological characteristics of apoptosis; and 2) the Ltx receptor is a beta2 integrin, a molecule which when stimulated is capable of elevating cytosolic 2+Ca levels.
In Specific Aim #1, Ltx-treated cells will be evaluated by DNA degradation, macromolecular synthesis, and FACS-associated morphologic alterations to determine if programmed cell death is occurring.
Specific Aim 2 will focus on the expression of genes known to be involved in programmed cell death such as Bc1-2, Bc1-x, ICE, Bax, CPP32 and p53 to determine a molecular mechanism for cell death.
Specific Aim 3 proposes experiments to define the nature of the toxin/receptor interaction. We envision that the first contact that the Ltx contact on a cell is a (32 integrin cell surface molecule. It is our goal to define these earliest consequences of this interaction such as determine the signal 2+pathway that is activated and the relationship to elevated cytosolic Ca +2 levels. Finally Specific Aim 4 will identify genes which are unique to Ltx- mediated apoptosis. It now emerging that the process of apoptosis involves different pathways when different agents are used to initiate the process. From these experiments we will have a better understanding of the pathway that is utilized by the toxin to kill cells. These studies provide fundamental insights into the pathobiology of the Aa Ltx and will bring us closer to understanding of its role in human periodontal infections on a molecular level. In addition, detailed studies on the action of the Aa Ltx will contribute to a better understanding of the biology of other membrane-active bacterial cytolynsins.
