Aggregatibacter actinomycetemcomitans leukotoxin (LtxA; Leukothera) induces cofilin dephosphorylation and actin depolymerization during killing of malignant monocytes

Abstract

Leukotoxin (LtxA; Leukothera), a protein toxin secreted by the oral bacterium Aggregatibacter actinomycetemcomitans, specifically kills white blood cells (WBCs). LtxA binds to the receptor known as lymphocyte function associated antigen-1 (LFA-1), a β2 integrin expressed only on the surface of WBCs. LtxA is being studied as a virulence factor that helps A. actinomycetemcomitans evade host defences and as a potential therapeutic agent for the treatment of WBC diseases. LtxA-mediated cell death in monocytes involves both caspases and lysosomes; however, the signalling proteins that regulate and mediate cell death remain largely unknown. We used a 2D-gel proteomics approach to analyse the global protein expression changes that occur in response to LtxA. This approach identified the protein cofilin, which underwent dephosphorylation upon LtxA treatment. Cofilin is a ubiquitous actin-binding protein known to regulate actin dynamics and is regulated by LIM kinase (LIMK)-mediated phosphorylation. LtxA-mediated cofilin dephosphorylation was dependent on LFA-1 and cofilin dephosphorylation did not occur when LFA-1 bound to its natural ligand, ICAM-1. Treatment of cells with an inhibitor of LIMK (LIMKi) also led to cofilin dephosphorylation and enhanced killing by LtxA. This enhanced sensitivity to LtxA coincided with an increase in lysosomal disruption, and an increase in LFA-1 surface expression and clustering. Both LIMKi and LtxA treatment also induced actin depolymerization, which could play a role in trafficking and surface distribution of LFA-1. We propose a model in which LtxA-mediated cofilin dephosphorylation leads to actin depolymerization, LFA-1 overexpression/clustering, and enhanced lysosomal-mediated cell death.

Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss

“…In particular, Itgal encodes CD11a, which is an integrin component of LFA-1. LFA-1 is considered a receptor for A. actinomycetemcomitans leukotoxin and is required for A. actinomycetemcomitans internalization, which initiates cell death (27). Since MK2 deficiency downregulated Itgal, the decreases in the amount of macrophage infiltrate seen were not likely due to A. actinomycetemcomitans-induced monocyte apoptosis.…”

Structure and function of RTX toxins

“…In addition, some RTX toxins can be engineered for therapeutic applications. The A. actinomycetemcomitans LtxA leukotoxin is being developed as a targeting agent (Leukothera®) for the treatment of hematological malignancies or autoimmune/inflammatory diseases, characterized by chronic activation of leukocytes expressing high levels of LFA-1 [387][388][389][390]. The B. pertussis CyaA toxin is used as an antigen delivery vehicle to trigger cell-mediated immune responses against a variety of antigens [379,391,392].…”

LFA-1-targeting Leukotoxin (LtxA; Leukothera®) causes lymphoma tumor regression in a humanized mouse model and requires caspase-8 and Fas to kill malignant lymphocytes

“…Others have reported that caspases play an important role in LtxA-mediated death of lymphocytes [11, 14, 15, 17, 26]. Therefore, to begin to decipher the mechanism of LtxA-mediated death of malignant lymphocytes, we examined the role of caspases.…”

“…After binding to LFA-1, LtxA induces the uptake of the LFA-1/LtxA complex and delivery to the lysosome, where the lysosomal membrane is disrupted and cathepsin D is released [11]. This mechanism of cell death in monocytes is very rapid and irreversible also resulting in cofilin dephosphorylation and actin depolymerization [15]. However, we found that lymphocytes do not undergo a similar mechanism of cell death since inhibitors of the lysosomal pathway do not affect LtxA-mediated cell death in these cells [11].…”