Aggregatibacter Actinomycetemcomitans Leukotoxin (Ltxa) Requires Death Receptor Fas, In Addition To Lfa-1, To Trigger Cell Death In T Lymphocytes

Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacterium Aggregatibacter actinomycetemcomitans. A. actinomycetemcomitans is an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans by binding to the beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. In addition, because of its specificity for malignant and activated WBCs, LtxA is being investigated as a therapeutic agent for treatment of hematological malignancies and autoimmune diseases. Here, we report the successful generation and characterization of Jurkat T lymphocytes with deletions in CD18, CD1 la, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LEA-1. We also demonstrate the requirement of the cell death receptor Fas for LtxA-mediated cell death in T lymphocytes. We show that LFA-1 and Fas are early events in the LtxA-mediated cell death cascade as caspase activation and mitochondrial perturbation do not occur in the absence of either receptor. To our knowledge, LtxA is the first molecule, other than FasL, known to require the Fas death receptor to initiate cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the understanding of LtxA as a bacterial virulence factor and development of it as a potential therapeutic agent.