Fusobacterium necrophorum leukotoxin induces activation and apoptosis of bovine leukocytes

Abstract

Fusobacterium necrophorum, a gram-negative, rod-shaped, anaerobic bacterium, is a primary or secondary etiological agent in a variety of necrotic, purulent infections in humans and animals. Its major virulence factor is leukotoxin, a high-molecular-weight secreted protein, primarily toxic to ruminant leukocytes. In this study, bovine peripheral blood leukocytes were exposed to various concentrations of immunoaffinity-purified leukotoxin and the cytotoxicity was analyzed by flow cytometry and scanning and transmission electron microscopy. At very low toxin concentrations, polymorphonuclear leukocytes (PMNs) showed activation, as indicated by translocation of primary and secondary granules to the periphery of the cytoplasm. Furthermore, these cells showed changes characteristic of apoptosis, including decreased cell size, organelle condensation, cytoplasmic membrane blebbing (zeiosis), and chromatin condensation and margination, and decrease in cellular DNA content. At moderately high concentrations of leukotoxin, bovine mononuclear cells were also induced to undergo programmed cell death. At very high concentrations, leukotoxin caused necrotic cell death of bovine peripheral leukocytes. The ability of F. necrophorum leukotoxin to modulate the host immune system by its toxicity, including cellular activation of PMNs and apoptosis-mediated killing of phagocytes and immune effector cells, represents a potentially important mechanism of its pathogenesis.

FIG. 1.

Flow cytometry scatter plot analysis of untreated peripheral blood leukocytes (A) and leukocytes treated with 20 (B) or 200 U of leukotoxin/ml (C). The upper gated region (R1) represents granulocytes, whereas the lower gated region (R2) includes PBMC. FSC-H (x axis), fluorescence channels for FSC of cells (proportional to size); SSC-H (y axis), fluorescence channels for orthogonal light scatter of cells (proportional to granularity).

FIG. 2.

Two-color dot plot analysis of simultaneous phagocytosis and oxidative burst activity of untreated granulocytes (A) and granulocytes treated with 40 U of leukotoxin/ml (B). Lower left quadrant, cells negative for phagocytosis and ROI; upper left quadrant, cells with phagocytosed PI-labeled S. aureus, but negative for ROI; lower right quadrant, cells reactive for ROI but negative for phagocytosis; upper right quadrant, cells with simultaneous phagocytosis and ROI.

FIG. 3.

SEM of leukotoxin-treated and untreated bovine peripheral blood leukocytes. Untreated cells appear single with minimal clumping (A and B). (B) Normal PMNs and mononuclear cells. At low concentrations of leukotoxin, PMNs showed decrease in cell size and clumping (C and D). Few cells at 0.02 to 2 U of toxin/ml showed surface lesions resembling pores (E) or small craters (F). At 20 U of leukotoxin/ml, huge craters appeared on the surfaces of PMNs (G). At high concentration (over 200 U/ml) cells agglutinated (H) and failed to separate even after DNase treatment (I).

FIG. 4.

Apoptotic changes of PMNs (A to D) and lymphocytes (E and F). Plasma membrane blebbing (bl; B to E) and apoptotic bodies (APB; A and F) are shown. Leukotoxin concentrations, 2 (A to C) and 20 U/ml (D to F).

FIG. 5.

TEM of bovine peripheral leukocytes treated with low concentrations of leukotoxin. Normal PMNs (P) and lymphocytes (L) appeared among untreated cells (A). Chromatin condensation was noticed in single or multiple nuclei (n), marginated as rounded or crescent-shaped structures in monocytes (B) and neutrophils (C and D). Apoptotic bodies (APB) were found with their characteristic condensed nuclei surrounded by a thin cytoplasm (E). Cytoplasm of phagocytes showed peripheral translocation of granules (gr; H) and vacuolation (F and G), with vacuoles (v) sometimes appearing large and containing material similar to the extracellular contents (I). (A) Untreated cells; (B to C and F to H) cells treated with 0.2 U of leukotoxin/ml; (D) cells treated with 0.02 U/ml; (E and I) cells treated with 20 U/ml. Bars (all panels), 1 μm.

FIG. 6.

Cytoplasmic organelles from cells undergoing apoptosis versus cells undergoing necrosis. Mitochondria (m) and endoplasmic reticulums (er) appeared as condensed, electron-dense structures with discernible internal architecture (A to D), whereas mitochondria in cells undergoing necrosis appeared as light-staining, swollen structures with minimal internal structures (E and F). (A to C) Cells treated with 2 U of leukotoxin/ml; (D) cells treated with 20 U/ml; (E) cells treated with 625 U/ml; (F) cells treated with 2,000 U/ml. Bars (all panels), 1 μm.

FIG. 7.

TEM of bovine peripheral lymphocytes treated with high concentrations of leukotoxin. Cells show perinuclear vacuolation (A) and various stages of necrosis (B, D, and E) or apoptosis (C and F). Lymphocytes appeared normal (L) or as apoptotic bodies (APB) in a milieu made of cytoplasmic contents from dead cells. (A) Cells treated with 200 U of leukotoxin/ml; (B to D and F) cells treated with 625 U/ml; (E) cells treated with 1,250 U/ml. Bars (all panels), 3 μm.

FIG. 8.

LDH activity of bovine leukocytes untreated (cells only) or treated with Triton X-100 (positive control) or leukotoxin at various concentrations (200,000 to 0.0005 U/ml).