Sodium polyanethole sulfonate as an inhibitor of activation of complement function in blood culture systems

Abstract

Sodium polyanethole sulfonate (SPS; trade name, Liquoid) is a constituent in culture media used to grow bacteria from blood samples from patients suspected of bacteremia. SPS prevents the killing of bacteria by innate cellular and humoral factors. We analyzed the effect of SPS on the three complement activation pathways: the classical, alternative, and lectin pathways, respectively. Inhibition of complement activity by SPS is caused by a blocking of complement activation and is not a result of complement consumption. The classical pathway is inhibited at SPS concentrations greater than 0.1 mg/ml, and complete inhibition is seen at 0.4 mg/ml. An SPS concentration of 0.5 mg/ml completely inhibits the binding of C1q and subsequent incorporation of C3, C4, and C9. The same was observed for the alternative pathway with an inhibition at SPS concentrations from 0.1 mg/ml and a complete inhibition from 0.4 mg/ml. Here, properdin binding was completely absent, and no incorporation of C3 and C9 was observed. In contrast, the lectin complement pathway remains unaffected at these SPS concentrations, and inhibition is first observed from 0.7 mg/ml. A complete inhibition required concentrations greater than 1 mg/ml. SPS is used in growth media (e.g., BACTEC and BacT/Alert) at concentrations from 0.3 to 0.5 mg/ml. The well-known finding that certain bacteria are growth inhibited by blood factors could therefore be a consequence of the lectin pathway, which is not inhibited at these concentrations. In addition, our findings also open up the possibility of a new assay for the assessment of the functional capacity of the lectin complement pathway.

FIG. 1.

(A) C3c levels in serum samples measured by a C3c-specific sandwich ELISA. Serum was incubated with inulin, zymosan, aggregated IgG (HA IgG), CVF, or SPS and control serum without application. After incubation, sera were made 10 mM with respect to EDTA, diluted 1:320. The C3c levels were assessed with a C3c-specific MAb (F1-4) as catching antibody and levels were measured as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations. (B) Western blot of human serum activated with inulin, zymosan (Zymo), aggregated IgG (HA IgG), CVF, or SPS and control serum without application. Nonreducing SDS-PAGE of serum samples diluted 1:40. C3 bands were visualized with the anti-human C3 monoclonal antibody (HAV3-4). We observed three bands with apparent molecular sizes of 180, 175, and 140 kDa corresponding to native C3, C3b/iC3b, and C3c, respectively. The MW marker was Precision Plus Protein Prestained (Bio-Rad).

FIG. 2.

Incorporation of C3 on surface-bound activators of the three described complement activation pathways: CP, AP, and LP. pNHS was diluted 1:1 in dilution buffer in the presence of final SPS concentrations from 0 to 1 mg/ml. Afterward, the samples were further diluted 1:5 equivalent to a final serum concentration of 10%, and assays were carried out as described in Materials and Methods. Incorporation of C3 was determined as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations.

FIG. 3.

(A) Classical pathway. Twofold dilutions of pNHS or MBL-deficient serum in the presence or absence of SPS (final concentration of 0.5 mg/ml) incubated on an immune complex surface. The binding, activation, and deposition of C components C1q, C4, C3, and C9 were determined as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations. (B) Alternative pathway. Twofold dilutions of pNHS or C2-deficient serum in the presence or absence of SPS (final concentration, 0.5 mg/ml) incubated on an LPS surface with Mg²⁺-EGTA binding, activation, and the deposition of C3, properdin, and C9 was assessed by using specific MAbs and determined as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations. (C) Lectin pathway. Twofold dilutions of pNHS or MBL-deficient serum in the presence or absence of SPS (final concentration, 0.5 mg/ml) incubated on a mannan surface. Binding, activation, and deposition of MBL, C3, C4, and C9 were assessed by using specific MAbs and determined as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations.

FIG. 3.

(A) Classical pathway. Twofold dilutions of pNHS or MBL-deficient serum in the presence or absence of SPS (final concentration of 0.5 mg/ml) incubated on an immune complex surface. The binding, activation, and deposition of C components C1q, C4, C3, and C9 were determined as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations. (B) Alternative pathway. Twofold dilutions of pNHS or C2-deficient serum in the presence or absence of SPS (final concentration, 0.5 mg/ml) incubated on an LPS surface with Mg²⁺-EGTA binding, activation, and the deposition of C3, properdin, and C9 was assessed by using specific MAbs and determined as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations. (C) Lectin pathway. Twofold dilutions of pNHS or MBL-deficient serum in the presence or absence of SPS (final concentration, 0.5 mg/ml) incubated on a mannan surface. Binding, activation, and deposition of MBL, C3, C4, and C9 were assessed by using specific MAbs and determined as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations.

FIG. 4.

(A) Effect of increasing concentration of Ca²⁺ on the C1q binding in pNHS. (B) Effect of increasing concentration of Mg²⁺ on the C3 deposition in pNHS. In both experiments, pNHS was diluted 1:1 in the presence of a final SPS concentration of 0.5 mg/liter. The samples were assayed at 10% pNHS and carried out as described in Materials and Methods. Controls were pNHS without SPS application. The C3 deposition was determined as OD_490-650 units. Error bars indicate two times the standard deviation of double determinations.

FIG. 5.

Complement activity after incubation of pNHS with the growth media BacT/Alert (A) and BACTEC (B). One volume of pNHS was added to two volumes of growth medium, followed by assessment of the complement activity through the three complement pathway assays as described in Materials and Methods. The efficient concentration of SPS corresponds to ∼0.35 mg/ml in the final dilution.