Streptococcal pyrogenic exotoxin B induces apoptosis and reduces phagocytic activity in U937 cells

Abstract

Treatment of U937 human monocyte-like cells with Streptococcus pyogenes led to an induction of apoptosis in these cells. A comparison between the wild-type strain and its isogenic protease-negative mutant indicated that the production of streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, caused a greater extent of apoptosis in U937 cells. Further study using purified SPE B showed that this protease alone could induce U937 cells to undergo apoptosis, which was characterized by morphologic changes, DNA fragmentation laddering on the gel, and an increase in the percentages of hypodiploid cells. The protease activity of SPE B was required for apoptosis to proceed, since treatment with cysteine protease inhibitor E64 or heat inactivation abrogated this death-inducing effect. The SPE B-induced apoptosis pathway was interleukin-1beta converting enzyme (ICE) family protease dependent. Further experiments showed that the phagocytic activity of U937 cells was reduced by SPE B. Treatment with E64 and heat inactivation both abrogated this phagocytosis-inhibitory effect. Taken together, the present data show that SPE B not only possesses the ability to induce apoptosis in monocytic cells but also helps bacteria to resist phagocytosis by host cells.

FIG. 1

Induction of apoptosis by S. pyogenes NZ131 and its speB mutant SW510 in U937 cells. U937 cells were infected with NZ131 or SW510 for 2 h at an MOI of 40:1. After cultivation for an additional 22 h, the percentages of apoptotic cells, shown by the presence of hypodiploid cells, were determined by propidium iodide staining followed by flow cytometric analysis (A). The percentages of apoptosis at the MOI of 40:1, 20:1, and 10:1 are shown in panel B. Data are shown as the averages ± SD for duplicate wells. ∗∗, P < 0.01 by comparing the NZ131-treated group with the SW510-treated group.

FIG. 2

SPE B-induced apoptosis in U937 cells. U937 cells were cultured with various doses of purified SPE B for 24 h (A) and 48 h (B). The percentages of apoptotic cells were determined by propidium iodide staining followed by flow cytometric analysis. Data are shown as the averages ± SD for duplicate wells. ∗, P < 0.05 by comparing the SPE B-treated group with the nontreated group.

FIG. 3

Characteristics of apoptosis in SPE B-treated U937 cells. U937 cells were cultured in medium alone (A) or with 25 μg of SPE B per well (B), and the morphologic changes were observed after 24 h by staining with Liu’s solution. The apoptotic cells are indicated with arrows. DNA fragmentation was detected by agarose gel electrophoresis 18 h after culture in medium alone or with SPE B (C).

FIG. 4

Effects of heat inactivation and cysteine protease inhibitors on SPE B-induced U937 cell apoptosis. (A) U937 cells were cultured in medium alone, with SPE B, with heat-inactivated SPE B (56°C, 20 to 30 min) (#) or with SPE B plus E64. The percentages of apoptotic cells were determined after 48 h by propidium iodide staining followed by flow cytometric analysis. (B) U937 cells were cultured in medium alone or with SPE B in the presence or absence of ICE family protease inhibitors, and the percentages of apoptotic cells were determined after 24 h. Data are shown as the averages ± SD for duplicate wells.

FIG. 5

Effect of SPE B on phagocytic activity of U937 cells. (A) U937 cells were mixed with FITC-labeled beads at a ratio of 1:100 in the presence of various doses of SPE B, and the binding of fluorescent beads was assessed by flow cytometric analysis after 2 h. (B) U937 cells were mixed with FITC-labeled beads in the presence of SPE B, heat-inactivated SPE B (#), or SPE B plus E64. The binding of fluorescent beads was assessed, and the percentages of inhibition were determined. Data are shown as the averages ± SD for duplicate samples. ∗, P < 0.05; ∗∗, P < 0.01 (by comparing the SPE B-treated group with the nontreated group).