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 β2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. ABSTRACT 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 β2 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, 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.

[1]  Angela C. Brown,et al.  Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles , 2018, Toxins.

[2]  N. Balashova,et al.  Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells. , 2016, Molecular oral microbiology.

[3]  J. Bertino,et al.  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. , 2015, Leukemia research.

[4]  S. Kachlany,et al.  Expression and targeting of lymphocyte function‐associated antigen 1 (LFA‐1) on white blood cells for treatment of allergic asthma , 2015, Journal of leukocyte biology.

[5]  S. Kachlany,et al.  Leukotoxin kills rodent WBC by targeting leukocyte function associated antigen 1. , 2013, Comparative medicine.

[6]  S. Kachlany,et al.  Leukotoxin (Leukothera®) Targets Active Leukocyte Function Antigen-1 (LFA-1) Protein and Triggers a Lysosomal Mediated Cell Death Pathway* , 2012, The Journal of Biological Chemistry.

[7]  S. Kachlany,et al.  In vitro synergism between LFA-1 targeting leukotoxin (Leukothera™) and standard chemotherapeutic agents in leukemia cells. , 2011, Leukemia research.

[8]  C. Rosada,et al.  Resolution of psoriasis by a leukocyte-targeting bacterial protein in a humanized mouse model. , 2011, The Journal of investigative dermatology.

[9]  Michael Loran Dustin,et al.  HIV Envelope gp120 Activates LFA-1 on CD4 T-Lymphocytes and Increases Cell Susceptibility to LFA-1-Targeting Leukotoxin (LtxA) , 2011, PloS one.

[10]  D. Speicher,et al.  Aggregatibacter actinomycetemcomitans leukotoxin is post-translationally modified by addition of either saturated or hydroxylated fatty acyl chains. , 2011, Molecular oral microbiology.

[11]  A. Sjöstedt,et al.  Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin , 2011, Cell Death and Disease.

[12]  David A. Cheresh,et al.  Integrins in cancer: biological implications and therapeutic opportunities , 2010, Nature Reviews Cancer.

[13]  N. Balashova,et al.  Anti-leukemia activity of a bacterial toxin with natural specificity for LFA-1 on white blood cells. , 2010, Leukemia research.

[14]  S. Kachlany,et al.  Aggregatibacter actinomycetemcomitans Leukotoxin , 2010, Journal of dental research.

[15]  N. Balashova,et al.  Aggregatibacter actinomycetemcomitans LtxC is required for leukotoxin activity and initial interaction between toxin and host cells. , 2009, Gene.

[16]  D. Speicher,et al.  Aggregatibacter actinomycetemcomitans leukotoxin requires β‐sheets 1 and 2 of the human CD11a β‐propeller for cytotoxicity , 2007, Cellular microbiology.

[17]  N. Balashova,et al.  Human CD18 Is the Functional Receptor for Aggregatibacter actinomycetemcomitans Leukotoxin , 2007, Infection and Immunity.

[18]  D. Speicher,et al.  Aggregatibacter actinomycetemcomitans leukotoxin requires beta-sheets 1 and 2 of the human CD11a beta-propeller for cytotoxicity. , 2007, Cellular microbiology.

[19]  E. Lally,et al.  Actinobacillus actinomycetemcomitans leukotoxin requires lipid microdomains for target cell cytotoxicity , 2006, Cellular microbiology.

[20]  N. Balashova,et al.  Characterization of leukotoxin from a clinical strain of Actinobacillus actinomycetemcomitans. , 2006, Microbial pathogenesis.

[21]  T. Kinashi,et al.  Intracellular signalling controlling integrin activation in lymphocytes , 2005, Nature Reviews Immunology.

[22]  N. Hogg,et al.  How T cells use LFA-1 to attach and migrate. , 2004, Immunology letters.

[23]  R. Claesson,et al.  Caspase 1 Involvement in Human Monocyte Lysis Induced by Actinobacillus actinomycetemcomitans Leukotoxin , 2003, Infection and Immunity.

[24]  J. Moyano,et al.  Engagement of α4β1 integrin by fibronectin induces in vitro resistance of B chronic lymphocytic leukemia cells to fludarabine , 2002, Journal of Leukocyte Biology.

[25]  P. Howard,et al.  Maintenance of oxidative phosphorylation protects cells from Actinobacillus actinomycetemcomitans leukotoxin‐induced apoptosis , 2001, Cellular microbiology.

[26]  JS Damiano,et al.  Cell adhesion-mediated drug resistance (CAM-DR) protects the K562 chronic myelogenous leukemia cell line from apoptosis induced by BCR/ABL inhibition, cytotoxic drugs, and γ-irradiation , 2001, Leukemia.

[27]  J. Korostoff,et al.  Perturbation of mitochondrial structure and function plays a central role in Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis. , 2000, Microbial pathogenesis.

[28]  I. Caragol,et al.  A novel CD18 genomic deletion in a patient with severe leucocyte adhesion deficiency: a possible CD2/lymphocyte function‐associated antigen‐1 functional association in humans , 2000, Immunology.

[29]  S. Dirnhofer,et al.  Expression of LFA-1 identifies different prognostic subgroups in patients with advanced follicle center lymphoma (FCL). , 1999, Leukemia research : a Forum for Studies on Leukemia and Normal Hemopoiesis.

[30]  P. Stanley,et al.  Acylation of Escherichia coli Hemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function , 1998, Microbiology and Molecular Biology Reviews.

[31]  M. Peter,et al.  Two CD95 (APO‐1/Fas) signaling pathways , 1998, The EMBO journal.

[32]  Jian Fei Wang,et al.  RTX Toxins Recognize a β2 Integrin on the Surface of Human Target Cells* , 1997, The Journal of Biological Chemistry.

[33]  K. Elkon,et al.  Requirement of Cysteine-rich Repeats of the Fas Receptor for Binding by the Fas Ligand* , 1997, The Journal of Biological Chemistry.

[34]  Manuel C. Peitsch,et al.  Characterization of Fas (Apo-1, CD95)-Fas Ligand Interaction* , 1997, The Journal of Biological Chemistry.

[35]  T. Springer,et al.  Characterization of lymphocyte function-associated antigen 1 (LFA-1)-deficient T cell lines: the alphaL and beta2 subunits are interdependent for cell surface expression. , 1997, Journal of immunology.

[36]  P. Kiener,et al.  Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils , 1996, The Journal of experimental medicine.

[37]  M. Peter,et al.  Cytotoxicity‐dependent APO‐1 (Fas/CD95)‐associated proteins form a death‐inducing signaling complex (DISC) with the receptor. , 1995, The EMBO journal.

[38]  P. Stanley,et al.  Fatty acylation of two internal lysine residues required for the toxic activity of Escherichia coli hemolysin. , 1994, Science.

[39]  N. Hogg,et al.  Control of leukocyte integrin activation. , 1993, The American review of respiratory disease.

[40]  A. Fischer,et al.  Leukocyte adhesion deficiency: molecular basis and functional consequences. , 1988, Immunodeficiency reviews.

[41]  G. Inghirami,et al.  Differential expression of LFA-1 molecules in non-Hodgkin's lymphoma and lymphoid leukemia. , 1988, Blood.