Abstract
Leukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca2+, activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X7R antagonist. Similarly, blockage of P2X7Rs with oATP prevented the intracellular mobilization of Ca2+, an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X7R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death.
Highlights
Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a Gram-negative oral bacterium found in the oral flora of healthy individuals and patients with aggressive periodontitis
The expelled ATP and activation of P2X7R allows the cell to proceed with the mobilization of intracellular Ca2+, caspase activation, and PARP cleavage, all of which are key events required for the LtxA-mediated cell death of Jurkat cells
Knowing that LtxA triggers caspase activation and subsequent Fas mediated cell death in Jurkat cells, we investigated whether Panx[1] channels were responsible for the release of ATP and if this contributed to the cell death pathway(s) that we observed
Summary
Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a Gram-negative oral bacterium found in the oral flora of healthy individuals and patients with aggressive periodontitis. Activation of LFA-1 plays a role in the migration of immune cells to the appropriate tissue in order to establish an inflammatory response and promote diapedesis, as the ability of immune cells to penetrate into tissues depends on activated LFA-16,7. LtxA initiates a lysosomal mediated death pathway where LtxA delivers LFA-1 to lysosomes, disrupting lysosomal integrity and inducing cell d eath[17] This was determined via the cytosolic detection of lysosomal-associated membrane protein 1 (LAMP-1) and cathepsin D following LtxA treatment, an indication of lysosomal lysis. When this pathway was studied in a lymphocytic leukemia cell model, this lysosomal mediated death mechanism was not observed[4]. The expelled ATP and activation of P2X7R allows the cell to proceed with the mobilization of intracellular Ca2+, caspase activation, and PARP cleavage, all of which are key events required for the LtxA-mediated cell death of Jurkat cells
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