Application of flow cytometry in toxinology: Pathophysiology of human polymorphonuclear leukocytes damaged by a pore‐forming toxin from Staphylococcus aureus

Abstract

The pore-forming activity of leukocidin (PVL) secreted by Staphylococcus aureus has been investigated on human white cells by flow cytometry techniques. This two-component toxin induced morphological modifications of neutrophils and monocytes as detected by forward light scattering measurements, but was inactive on lymphocytes. These modifications were the consequence of pore formation through the cell membrane leading to its permeabilization. In the absence of calcium, PVL formed pores large enough to allow ethidium ions to penetrate the cells and become fluorescent by intercalating nucleic acids. In the presence of calcium, the pores were too small for ethidium entry but Key terms: Staphylococcus aureus, polymorphonuclear neutrophil, monocyte, HL-60, leukocidin, pore-forming toxin, cell activation, flow cytometry Many bacterial toxins provoke lesions in the plasma membrane and modify its permeability to ions or even to proteins. These toxins are called "pore-forming toxins" and 102 have been isolated and characterized to date ( 1). Among these, several are synthesized and excreted by Staphylococcus aureus: alpha-toxin (2), delta toxin (10) and synergohymenotropic toxins (1 2). Amid the latter, the so-called Panton-Valentine leukocidin (PVL) consisting of two synergic proteins S (32,000 da) and F (38,000 da) has been purified (8) and sequenced (12). The PVL is toxic for polymorphonuclear neutrophils (PMNs) but is not hemolytic. Pore formation requires the presence of the two components of the toxin and the events sequence consists of the initial binding of S followed by binding of F ( 5 ) . The pores formed by the toxin through the membrane of PMNs are ion-sized in the presence of extracellular divalent cations which enter the cells (7). When calcium is present, the calcium influx is responsible for the secretion of the granule contents of PMNs (5).The aim of the present study was to show that flow cytometry is a suitable technique to obtain a simultaneous evaluation of these different parameters and determine the cell specificity of PVL. Moreover, flow cytometry allowed us to determine the morphological modifications of the PMNs consecutive to the membrane permeability variations appearing after PVL binding. MATERIALS AND METHODS Toxin ProductionThe two components S and F of the PVL were purified, as described previously (S), by chromatography of the supernatant of S aureu V8 strain (ATCC 49775). Preparation of White CellsWhite cells were prepared from buffy-coats of healthy donors, or from blood collected from patients with This investigation was supported in part by a grant (CRE 900316) from the

The Bicomponent Pore-Forming Leucocidins of Staphylococcus aureus

“…In this work, it was proposed that ethidium entry into host cells, but not calcium entry, is a definitive marker of pore formation by leucocidins (152,153). In contrast, calcium entry upon leucocidin treatment is believed to occur prior to pore formation and is mediated by the opening of membrane divalent cation channels (154,155).…”

“…This evidence is as follows: (i) PVL has a more limited host range than LukED (it is not lytic on murine immune cells); (ii) PVL exhibits a more restricted immune cell-targeting profile, as it kills primarily neutrophils, monocytes, and macrophages; (iii) of the leucocidin subunits, only HlgC is capable of competing with fluorescently labeled LukS-PV for binding of the surface of primary leukocytes; and (iv) the most divergent regions of PVL and LukED occur in the rim domain of the protein believed to be most critical for receptor recognition and cellular specificity ( Fig. 3 and 5) (153,174,200,230). Interestingly, the ability of HlgC to successfully compete with labeled LukS-PV for cell surface binding suggests that PVL and HlgCB of gamma-hemolysin may target similar receptors on the cell surface, although this has not yet been validated experimentally (174).…”

Retrieving Biological Activity from LukF-PV Mutants Combined with Different S Components Implies Compatibility between the Stem Domains of These Staphylococcal Bicomponent Leucotoxins

“…Human PMNs from healthy donors were purified from buffy coats as previously reported (2) and resuspended at 5 ϫ 10 5 cells/ml. Flow cytometry measurements from 3,000 PMNs were performed using a FacSort cytometer (Becton Dickinson, Le Pont de Claix, France) equipped with an argon laser tuned at 488 nm (16). Fluo-3 fluorescence (Molecular Probes) for Ca 2ϩ entry and ethidium fluorescence were acquired with Lysis II software (Becton Dickinson) as previously mentioned (2).…”

Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils

“…This pore is an octameric β-barrel molecular complex (9) perpendicular to the plane of the cell membrane, similar to that made by S. aureus α-toxin (10). Sublytic concentrations of purified PVL induce pronounced histamine release from human basophils and stimulate human neutrophils to release enzymes (β-glucuronidase and lysozyme), chemotactic components (leukotriene-B4 and IL-8), and oxygen metabolites (10)(11)(12)(13)(14).…”