title: Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells.
abstract: Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8-7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng ml(-1) LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH-sensitive indicator (pHrodo(®)) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker(®) Red DND-99 staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells.
authors.affiliation: Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, PA, USA.
authors.forename: N
authors.initials: N
authors.lastname: Balashova
authors.affiliation: Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Dhingra
authors.affiliation: Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA, USA.
authors.forename: K
authors.initials: K
authors.lastname: Boesze-Battaglia
authors.affiliation: Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, PA, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Lally
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D004789:Enzyme Activation / Q000187:drug effects; D005098:Exotoxins / Q000494:pharmacology*; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020014:K562 Cells; D008051:Lipid Bilayers; D016169:Lymphocyte Function-Associated Antigen-1 / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism
chemical_list: D005098:Exotoxins; D008051:Lipid Bilayers; D016169:Lymphocyte Function-Associated Antigen-1; C018579:leukotoxin
keywords: RTX toxin; bacterial pathogenesis; cytosol acidification; cytosolic pH; lysosome