A new pathway of staphylococcal pathogenesis: apoptosis-like death induced by Staphopain B in human neutrophils and monocytes

Abstract

Circulating neutrophils and monocytes form the first line of cellular defense against invading bacteria. Here, we describe a novel and specific mechanism of disabling and eliminating phagocytes by Staphylococcus aureus. Staphopain B (SspB) selectively cleaved CD11b on phagocytes, which rapidly acquired features of cell death. SspB-treated phagocytes expressed phosphatidylserine as well as annexin I and became permeable to propidium iodide, thus demonstrating distinctive features of both apoptosis and necrosis, respectively. The cell death observed was caspase and Syk tyrosine kinase independent, whilst cytochalasin D efficiently inhibited the staphopain-induced neutrophil killing. Neutrophil and monocyte cell death was not affected by integrin clustering ligands (ICAM-1 or fibrin) and was prevented, and even reversed, by IgG. This protective effect was dependent on the Fc fragment, collectively suggesting cooperation of the CD16 receptor and integrin Mac-1 (CD11b/CD18). We conclude that SspB, particularly in the presence of staphylococcal protein A, may reduce the number of functional phagocytes at infection sites, thus facilitating colonization and dissemination of S. aureus.

Fig. 1

SspB induces death in human neutrophils and monocytes in a proteolytic activity-dependent manner in the absence of human serum. Neutrophils (PMN) and monocytes (Mo) were treated with 100 nM SspB for 75 min at 37°C in the presence of 1% BSA and then washed with culture media to remove the enzyme. Control or treated cells were analyzed for externalization of PS and cell membrane permeability by staining with FITC-labeled AnxV and PI, respectively. Cells were analyzed by flow cytometry to determine the percentage of (1) healthy cells (AnxV–/PI–) negatively stained with both AnxV and PI, (2) cells expressing PS but with an intact plasma membrane (AnxV+/PI–), (3) cells with both externalized PS and a compromised membrane (AnxV+/PI+) and (4) cells without external PS but positively stained with PI (AnxV–/PI+). The percentage of cells in each subpopulation counted in quadrants is shown in the insets of representative dot plots (a and d). a Result of an experiment assessing the effect of active SspB (+SspB) on neutrophils (PMN) and monocytes (Mo). Bar graphs (b and c) represent mean percentage values ± SD of each cell subpopulation inferred from 7 experiments with neutrophils (b) and 3 with monocytes (c). * p < 0.05; ** p < 0.01; *** p < 0.001. d Effect of E-64-treated protease (+SspB + E-64) and the inactive recombinant form of the protease (+SspB C24A) on neutrophils.

Fig. 2

Apoptotic DNA fragmentation in neutrophils and monocytes following treatment with SspB. DNA isolated from neutrophils (PMN) and monocytes (Mo) cultured for 24 h in the presence of various concentrations of SspB (in medium with 1% BSA) was subjected to electrophoretic analysis. Lane 1 = control cells; lanes 2–6 = cells incubated with 12.5, 25, 50, 75 and 100 nM SspB, respectively; M = DNA size marker (O'GeneRuler^TM; Fermentas, Vilnius, Lithuania). Three reference bands correspond to 3, 1 and 0.5 kb. Control cells (freshly isolated from peripheral blood) were incubated in the same medium without the protease. The data are from 1 representative experiment of 5 performed.

Fig. 3

Presence of serum reverses SspB-induced exposure of PS and AnxI on the outer leaflet of neutrophil cytoplasmic membrane. Neutrophils were preincubated with 100 nM SspB, after which the enzyme was removed and cells were incubated for 2 and 12 h in medium with 2% human serum. Exposure of PS and AnxI was measured immediately (a and b), 2 h (c and d) and 12 h (e and f) after 70 min of treatment with SspB. Green represents fluorescence intensity of control neutrophils, red represents SspB-treated neutrophils, dark blue represents neutrophils incubated with dithiothreitol, an activation factor for SspB, pale blue represents spontaneously apoptotic neutrophils (aged 24 h) which served as a positive control for AnxI exposure and gray represents the autofluorescence of control cells. The results of 1 representative experiment of 3 performed are presented.

Fig. 4

SspB induces neutrophil death in a caspase- and Syk tyrosine kinase-independent manner via effects on the actin cytoskeleton. Neutrophils were preincubated in medium with 1% BSA for 15 min in the presence of zVAD-fmk (zV; 75 or 150 LIM), piceatannol (PIC; 20 LIM) and cytochalasin D (Cyt D; 100 LIM) prior to SspB incubation (100 nM, 75 min). Control or treated cells were stained with FITC-labeled AnxV and PI, followed by flow cytometry analysis to determine the percentage of AnxV-/PI-, AnxV+/PI-, AnxV+/PI+ and AnxV-/PI+ cells. For details, see legend of figure 1. Data are the means ± SD from 2 independent experiments.

Fig. 5

IgG and IgG-derived Fc fragments abolish SspB-induced death of neutrophils whilst SpA intensifies the effect of the protease, a Neutrophils were preincubated with IgG (11 mg/ml) or their Fc fragments (7 mg/ml) in medium with 1% BSA for 15 min prior to SspB treatment (100 nM, 75 min). NS = Not significant. b Cells were treated with SspB (100 nM, 75 min) in medium with 2% BSA, 2% human serum (HS), 2% protein A-Sepharose CL 4B-adsorbed human serum (HS PrA-SE) or 2% serum preincubated with Sepharose 4B (HS SE; unspecific binding control). Control or treated cells were stained with FITC-labeled AnxV and PI, followed by flow cytometry analysis to determine the percentage of AnxV–/PI–, AnxV+/PI–, AnxV+/PI+ and AnxV–/PI+ cells. For details, see legend of figure 1. Data are the means ± SD from 3 (a) and 2 (b) independent experiments.

Fig. 6

SspB decreases surface expression of CD16 on neutrophils (a) as well as CD11b/CD18 on both neutrophils and monocytes (b). a Neutrophils were incubated in medium with 1% BSA with 100 nM active SspB and the inactive recombinant form of the enzyme (C24A), with or without the addition of IgG. Control and treated cells were stained with anti-CD16 fluorescently labeled monoclonal antibody, followed by flow cytometry analysis. The dotted line represents fluorescence intensity of control cells, the thick black line represents neutrophils treated with 100 nM SspB, the dashed line represents cells treated with 100 nM SspB in the presence of IgG and the thin black line represents neutrophils incubated with inactive SspB (C24A). b Neutrophils (PMN) and adherent (Mo adh) as well as nonadherent monocytes (Mo) were incubated in medium with 1% BSA with either 100 nM active SspB or the inactive form of SspB, C24A (only for PMN), and stained using an anti-CD11b fluorescently labeled monoclonal antibody, followed by flow cytometry analysis. The dotted line represents fluorescence intensity of control cells, the thick black line represents cells treated with 100 nM SspB, the thin black line (only for PMN) represents cells incubated with inactive SspB (C24A). Light and dark gray areas represent autofluorescence of control and SspB-treated cells, respectively (a, b). The results of 1 representative experiment of 3 performed are presented.