Streptococcus suis interactions with the murine macrophage cell line J774: adhesion and cytotoxicity

Abstract

Streptococcus suis capsular type 2 is an important etiological agent of swine meningitis, and it is also a zoonotic agent. Since one hypothesis of the pathogenesis of S. suis infection is that bacteria enter the bloodstream and invade the meninges and other tissues in close association with mononuclear phagocytes, the objective of the present study was to evaluate the capacity of S. suis type 2 to adhere to macrophages. An enzyme-linked immunosorbent assay technique was standardized to simply and accurately measure the rate of bacterial attachment to phagocytic cells. Results were confirmed by plate counting. Adhesion was dependent on bacterial concentration and incubation time and was not affected by cytochalasin pretreatment of macrophages. Inhibition studies showed that the sialic acid moiety of the S. suis capsule would be, at least in part, responsible for bacterial recognition by macrophages. Serum preopsonization of bacteria increased adhesion levels. Complement would be partially implicated in the serum-enhanced binding of S. suis to cells. Adhesion varied among different S. suis type 2 isolates. However, high bacterial concentrations of several isolates were cytotoxic for cells, and these cytotoxic effects correlated with suilysin production. Indeed, hemolytic strain supernatants, as well as purified suilysin, reproduced cytotoxic effects observed with live bacteria, and these effects were inhibited by cholesterol pretreatment. Bacterial adhesion and cytotoxicity were confirmed by scanning and transmission electron microscopy. We hypothesize that attachment of bacteria to phagocytes could play an important role in the pathogenesis of S. suis infection by allowing bacterial dissemination and causing a bacteremia and/or septicemia. This interaction could also be related to the activation of the host inflammatory response observed during meningitis.

FIG. 1.

(A) Kinetics of S. suis adhesion to J774 macrophages with an MOI of 10 bacteria/cell (∼10⁶ CFU/well). The kinetics of inoculum multiplication during the adhesion assay is also shown. ∗, P < 0.001 with respect to 30-min adhesion levels; ∗∗, P < 0.01 with respect to values observed at other incubation times. (B) Effect of bacterial concentration on 30-min adhesion to J774 macrophages. Data are expressed as means ± standard deviations of bound bacteria (in number of CFU recovered per well).

FIG. 2.

Transmission electron micrographs showing S. suis adhesion to J774 macrophages at different parts of the plasma membrane. S. suis adhered to the plain cell surface (bar, 250 nm) (A) or to the cell surface projections (bar, 0.5 μm) (B). (C) Scanning electron micrograph showing a small chain of S. suis cocci adhering to the surface of macrophages (bar, 1 μm). Arrows indicate S. suis cocci.

FIG. 3.

Effect of bacterial preopsonization on 30-min adhesion to J774 macrophages. S. suis strain 31533 was preopsonized 30 min at 37°C with different concentrations of normal FBS, hiFBS, C'MS (C′), or heat-inactivated C'MS (hiC′). Preopsonized bacteria or nonopzonized control bacteria (bacteria preincubated 30 min with 5% BSA-2% dextrose in DMEM) were added at 10⁶ CFU/well to macrophage plates. Data are expressed as means ± standard deviations of percentage of increased adhesion with respect to adhesion found with control bacteria (100% adhesion).

FIG. 4.

Effect of incubation time in presence of S. suis strain 31533 on J774 injury. The cytotoxic effect of bacteria was evaluated in parallel by the LDH test and by the selective stain of cell nuclei of remaining macrophages with methylene blue (MB) after different intervals of bacterium-cell contact at an MOI of 100 bacteria/cell (10⁷ CFU/well). Data are expressed as the percentage of cytotoxicity (± standard deviations) in infected wells with respect to control wells with macrophages alone. ∗, P < 0.001 with respect to 30-min incubation time; ∗∗, P < 0.001 with respect to cytotoxic levels at 30 min and 1 h of incubation time.

FIG. 5.

Transmission electron micrographs demonstrating J774 macrophage injury after infection with 10⁸ CFU of S. suis per ml. (A) Noninfected control cells. (B) Cells incubated for 3 h with the suilysin-negative strain 89-1591. (C) Cells incubated with the suilysin-positive strain 31533. J774 integrity after 3 h of incubation with strain 89-1591 was comparable to that of noninfected control cells. Injury wasmanifested by the loss of cytoplasmic density, severe disruption of cytoplasmic membranes with release of cellular contents (arrowheads), and disappearance of the nucleus. Bars, 0.5 μm. Arrows indicate S. suis cocci.