Aggregation substance promotes adherence, phagocytosis, and intracellular survival of Enterococcus faecalis within human macrophages and suppresses respiratory burst

Abstract

The aggregation substance (AS) of Enterococcus faecalis, encoded on sex pheromone plasmids, is a surface-bound glycoprotein that mediates aggregation between bacteria thereby facilitating plasmid transfer. Sequencing of the pAD1-encoded Asa1 revealed that this surface protein contains two RGD motifs which are known to ligate integrins. Therefore, we investigated the influence of AS on the interaction of E. faecalis with human monocyte-derived macrophages which constitutively express beta(2) integrins (e.g., CD18). AS was found to cause a greater-than-fivefold increase in enterococcal adherence to macrophages and a greater-than-sevenfold increase in phagocytosis. Adherence was mediated by an interaction between the RGD motif and the integrin CD11b/CD18 (complement receptor type 3) as demonstrated by inhibition studies with monoclonal antibodies and RGD peptide. AS-bearing enterococci were significantly more resistant to macrophage killing during the first 3 h postinfection, probably due to inhibition of the respiratory burst as indicated by reduced concentrations of superoxide anion.

FIG. 1

Effect of bacterial concentration and time on adherence of E. faecalis to human macrophages. (A) Macrophages were incubated with various concentrations of enterococci at 37°C for 15 min. The number of FITC-labeled bacteria bound to 100 macrophages is expressed as the AI. Points represent mean values ± standard deviations of four independent assays with four wells. (B) Adherent macrophages were incubated with 2.5 × 10⁷ enterococci/ml for 15 to 60 min. Data represent means ± standard deviations of four independent assays with three wells. Symbols: ○, E. faecalis OG1X(pAM721) (AS positive); □, E. faecalis OG1X (AS negative).

FIG. 2

Adherence of E. faecalis mutants with various deletions in the structural gene asa1 in comparison to the AS-negative strain OG1X(pAM944) and the AS-positive strain OG1X(pAM944/pWHH6). Adherent macrophages were incubated with 2.5 × 10⁷ enterococci per ml for 15 min. Columns represent mean values ± standard deviations of four independent assays with three wells. ∗, P < 0.05 compared with OG1X(pAM944).

FIG. 3

Effect of RGDS on adherence of E. faecalis OG1X and OG1X(pAM721). Adherent macrophages were preincubated with various concentrations of peptide for 15 min prior to exposure to bacteria (2.5 × 10⁷ cells/ml) for 15 min. The 100% values without peptide are 98 ± 6 adherent bacteria per 100 macrophages for OG1X(pAM721) and 13 ± 1 for OG1X. Values are expressed as percentages of the mean value ± standard deviation from three independent experiments with three wells. Symbols: ○, OG1X(pAM721); □, OG1X.

FIG. 4

Inhibition of enterococcal attachment by pretreatment with anti-CD18 MAb IB4. Macrophages were preincubated with different concentrations of IB4 for 15 min at 37°C before bacteria were added. Data are expressed as percentages of the mean value ± standard deviation from three independent experiments with three wells. The 100% value represents adherent bacteria per 100 macrophages without addition of antibody and is 151 ± 10 for OG1X(pAM721) and 9 ± 0.2 for OG1X. Isotype-specific antibody which served as negative control had no effect (not shown). ○, OG1X(pAM721); □, OG1X.

FIG. 5

Inhibition of E. faecalis binding to macrophages by MAbs against CD11a (DF1524), CD11b (ICRF44), and CD11c (MAb 3.9). Macrophages were pretreated with MAbs for 15 min at 37°C prior to incubation with enterococcal strains OG1X and OG1X(pAM721). Data are expressed as mean values ± standard deviations (n = 3) of the AI described in the legend to Fig. 1. Open bars, OG1X; shaded bars, OG1X(pAM721). ∗, P < 0.05 versus control MAb IgG1 (W3/25).

FIG. 6

Respiratory burst activity of human macrophages in response to E. faecalis as measured by lucigenin-enhanced CL. Macrophages (5 × 10⁶/ml) were first incubated in medium containing lucigenin. At time zero, enterococci (5 × 10⁷/ml) were added and CL (light units) was measured at 2-min intervals over a time period of 120 min. Data are mean values ± standard deviations of three wells. The graphs show representative results of one of three experiments. Wells without bacteria served as negative controls. Control values ranged from 8 to 25 light units and were subtracted from test data. ○, AS-positive OG1X(pAM721); □, AS-negative OG1X.

FIG. 7

Time course of intracellular survival of AS-positive E. faecalis OG1X(pAM721) and AS-negative E. faecalis OG1X within human macrophages. Macrophages (2 × 10⁴) were allowed to ingest bacteria (2 × 10⁵) for 1 h. Extracellular bacteria were removed by washing and killed by penicillin and gentamicin. The results represent the mean ± standard deviation percent viable intracellular bacteria per 10⁵ macrophages of three independent experiments with two wells.