Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA) Requires Death Receptor Fas, in Addition to LFA-1, To Trigger Cell Death in T Lymphocytes

Abstract

Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacteriumAggregatibacter actinomycetemcomitans.A. actinomycetemcomitansis an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor forA. actinomycetemcomitansby binding to the β2integrin 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, CD11a, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LFA-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.

Aggregatibacter actinomycetemcomitans leukotoxin: From mechanism to targeted anti‐toxin therapeutics

“…The Jβ2.7 cell line is a mutant of the Jn.9 Jurkat T cell clone (Cherry, Weber, & Klickstein, 2001) that does not translate CD11a (Weber, York, Springer, & Klickstein, 1997). Although CD18 mRNA is present in the cell, evidence suggests that both subunits are required for cell surface expression of CD18 (Weber et al, 1997), a finding that was recently verified using knockout Jurkat E6.1 mutants (Vega et al, 2019). Thus, the Jβ2.7 cells express neither CD11a nor CD18; as a result, the cells are insensitive to LtxA (Kieba et al, 2007).…”

“…This specific targeting of host cells led to the hypothesis that LtxA acts in an immunosuppressive manner, to promote A. actinomycetemcomitans proliferation (Rabie et al, 1988). While this function has not yet been conclusively implicated in the etiology of LAP, significant additional evidence over the years has accumulated to indicate that LtxA specifically targets human immune cells (Balashova, Dhingra, Boesze‐Battaglia, & Lally, 2016; DiFranco et al, 2012; Kieba et al, 2007; Vega, Schober, Kim, Belinka, & Kachlany, 2019).…”

“…Knockouts in lymphocytes without CD18 were found to not be susceptible to LtxA, as were knockouts without CD11a (Vega et al, 2019). The authors noted that deletion of each individual subunit prevented cell surface expression of the other.…”

“…A similar requirement for both subunits in LFA‐1 function was reported previously (Weber et al, 1997). Because of this, Vega et al concluded that “[in] lymphocytes, both CD11a and CD18 are critical for LtxA to induce its toxic effects” (Vega et al, 2019).…”

“…The results discussed here have revealed the importance of both LFA‐1 subunits for LtxA activity, suggesting that there may be critical binding sites on both. Efforts to elucidate the location of the binding sites have been hampered by the observation that both subunits be present for either to be expressed on the surface of the cell (Vega et al, 2019). Consequently, any further study may require an extensive catalog of point mutations while being mindful of the impact the substitutions may have on the structural conformation of the proteins.…”