Streptococcal Inhibitor of Complement Inhibits Two Additional Components of the Mucosal Innate Immune System: Secretory Leukocyte Proteinase Inhibitor and Lysozyme

Bacterial strains.S. pyogenes type M1 strain NCTC 8198 (ATCC 12344) was obtained from the National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom. The sequence of its sic gene is identical to that originally published (GenBank accession no. X92968) (1, 11). The bacteria were grown in Todd-Hewitt broth-0.2% yeast extract (THBY) (Oxoid Ltd., Basingstoke, Hants., United Kingdom) and maintained short-term on selective Columbia agar-5% horse blood agar plates (HBA) (containing oxolinic acid at 5 μg/ml and colistin sulfate at 10 μg/ml; Sigma-Aldrich Co. Ltd., Gillingham, United Kingdom). To select for maximum expression of M protein and other virulence factors, the bacteria were passaged three times in fresh human plasma as described previously (3). Streptococcus suis type 2 strain P117, originally isolated from a pig with meningitis, was kindly provided by Andrew Allen, Bacterial Infection Group, Centre for Veterinary Science, University of Cambridge. The bacteria were grown in Todd-Hewitt broth and maintained on selective HBA plates as described above.

Purification of SIC.SIC was purified from 1-liter overnight-culture supernatants of strain M1 GAS, to which 10 mM EDTA had been added, as follows. A SIC-containing fraction was precipitated from the culture supernatant with 30% saturated solution of ammonium sulfate at room temperature for 1 h, the pH was adjusted to 5.5 with HCl, and the precipitate was spun down at 22,000 × g for 30 min at room temperature. The SIC precipitate was recovered from the sides of the tube by dissolving it in 50 mM phosphate-10 mM EDTA (pH 7) (PE), and this solution was made by adding solid ammonium sulfate to a final concentration of 0.4 M and applied to a 20-ml butyl Sepharose hydrophobic interaction chromatography (HIC) column (Amersham Pharmacia Biotech, Little Chalfont, Bucks., United Kingdom) equilibrated in the same buffer. The column was washed with PE-0.4 M ammonium sulfate, and the SIC was eluted with a 7-column-volume decreasing ammonium sulfate gradient from 0.4 down to 0 M in PE. SIC eluted as a clean prominent peak in the latter part of the gradient. We found that concentrating the fractions by ultrafiltration caused substantial loss of protein. Therefore, as a means of concentrating, the SIC was reprecipitated with 50% ammonium sulfate at room temperature and, to avoid any loss of protein, precipitation was carried out in the centrifuge tubes, because a significant proportion of the SIC precipitate sticks to the wall of any vessel used. The precipitate was spun down as before, redissolved in a minimal volume of phosphate-buffered saline (PBS), and dialyzed into PBS. Aliquots were stored at −70°C. Typically, 5 mg of SIC can be purified from 1 liter of culture supernatant by this procedure.

ELISA.Enzyme-linked immunosorbent assay (ELISA) plates (Microlon; Greiner Bio-One, Stroud, Glos., United Kingdom) were coated with either hen egg lysozyme (HEL), human lysozyme, lactoferrin from human milk (Sigma Aldrich Co. Ltd.), or recombinant human SLPI (rhSLPI) (R & D Systems Ltd., Abingdon, Oxford, United Kingdom) at 2 μg/ml in triplicate in 50 mM carbonate-bicarbonate buffer, pH 9.6 (or with coating buffer only as a control), and the plate was blocked with PBS-1% gelatin (VWR International Ltd., Lutterworth, United Kingdom). SIC at 10 μg/ml was added, and bound protein was detected with an in-house rabbit anti-SIC (immunoglobulin [Ig] fraction) (11) at 2 μg/ml followed by alkaline phosphatase-conjugated goat anti-rabbit Ig (Sigma Aldrich Co. Ltd.) diluted 1/1,500. All dilutions were in PBS-0.1% gelatin-0.05% Tween 20.

In other ELISAs, plates were coated with SIC at 1 μg/ml as described above and blocked with PBS-1% bovine serum albumin (BSA) or PBS-2.5% dried skim milk. Human lysozyme or lactoferrin was added at 5 μg/ml or HEL was added at 10 μg/ml, and bound protein was detected with sheep anti-human lysozyme (IgG fraction; CN Biosciences UK Ltd., Nottingham, United Kingdom) at 35 μg of total protein per ml, rabbit anti-human lactoferrin (Ig fraction; DAKO Ltd., Ely, United Kingdom) at 2.5 μg/ml, or rabbit anti-HEL (Ig fraction; Abcam Ltd., Cambridge, United Kingdom) at 5 μg/ml and then with alkaline phosphatase-conjugated monoclonal anti-sheep or -goat Ig (Sigma Aldrich Co. Ltd) diluted 1/1,500 or alkaline phosphatase-conjugated anti-rabbit Ig. All dilutions for the human lysozyme plate were in PBS-0.1% BSA-0.05% Tween 20, and those for the lactoferrin or HEL plate were in PBS-0.5% dried skim milk-0.05% Tween 20. In a similar ELISA, rhSLPI at 5 μg/ml was added to the SIC-coated plate and detected with goat anti-rhSLPI, the IgG fraction, at 1 μg/ml (R & D Systems Ltd.) and then with alkaline phosphatase-conjugated monoclonal anti-goat Ig as described above, with blocking and dilutions being in PBS-0.1% gelatin-0.05% Tween 20.

In all ELISAs, coating was at 4°C overnight, subsequent incubations were for 1 h at 37°C, and the final reagent was the Sigma Fast pNPP substrate-buffer system (Sigma Aldrich Co. Ltd.). One hundred-microliter volumes of all reagents were used throughout, except in the blocking step, for which reagents were at 200 μl. Absorbance was read at 405 nm (reference wavelength, 490 nm) in a model 3550 microplate reader (Bio-Rad Laboratories Ltd., Hemel Hempstead, Herts., United Kingdom). All ELISAs were performed at least three times with triplicate wells.

Iodination of proteins.Five hundred micrograms of HEL, 100 μg of rhSLPI, and 100 μg of lactoferrin were radiolabeled by the iodogen method (14) with 1 mCi of ¹²⁵I each (Amersham Pharmacia Biotech). Free iodine was removed by gel filtration on a PD10 column (Amersham Pharmacia Biotech). Specific activities of the labeled proteins were on the order of 10⁶ cpm/μg.

Interactions of SIC with SLPI, lysozyme, and lactoferrin binding studies.Double dilutions of rhSLPI or lactoferrin from 200 μg/ml (17.5 and 2.6 μM, respectively) at pH 7.3 in PBS-10 mM sodium azide-1 mg of BSA per ml, containing iodinated ligand to approximately 1.3 × 10⁴ cpm/μg, were added to SIC at 200 μg/ml (6.45 μM) and incubated overnight at 4, 22, or 37°C. Serial dilutions of HEL from 500 μg/ml (35 μM) containing the same proportion of labeled protein were combined with SIC at 100 μg/ml (3.22 μM) and incubated as described above. In all experiments complexes were precipitated with 1.5 M ammonium sulfate and centrifuged. The pellets and supernatants were counted in an LKB Wallac (Turku, Finland) 1277 Gamma Master automatic gamma counter. Results were analyzed by fitting data to the hyperbolic binding curve using nonlinear regression in order to obtain the K_d and number of ligand molecules bound. To make sure that we used only points where equilibrium had been reached, we made a preliminary plot of percentages of ligand bound versus amounts of ligand offered. All points where the percentage bound fell as ligand concentration decreased were rejected. Thermodynamic properties of the reactions were derived from the dissociation constants obtained at the three temperatures. Data were processed using the SPSS 10 statistical data analysis program (SPSS Inc., Chicago, Ill.). All binding experiments were performed at least three times. Due to the high cost of rhSLPI, the SLPI experiments were performed in duplicate, while the HEL and lactoferrin experiments were performed in triplicate.

Other binding studies.To investigate whether any breakdown of either protein occurred during incubation of SIC with lysozyme or SLPI, 5 μg of SIC was incubated overnight at 4°C with 5 μg of human lysozyme, HEL, or SLPI in 20-μl volumes. Samples (5 μg) of the individual proteins incubated alone served as controls. Reducing Laemmli loading buffer (20 μl) was added, and the samples were boiled for 3 min, subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (SDS-12.5% PAGE), and stained with Coomassie blue.

Antibacterial assays. (i) Titration of the effect of rhSLPI on M1 GAS.The effect of rhSLPI on the viability of the M1 (SIC-positive) strain of GAS was investigated by a method adapted from the work of Wiedow et al. (30). Overnight cultures of M1 GAS were grown at 37°C in THBY and used to inoculate fresh broth at a 1/100 dilution. Bacteria were grown to mid-log phase, spun down, washed twice in 10 mM sodium phosphate buffer (pH 7.2), and resuspended to a concentration of 2 × 10⁵/ml in phosphate buffer-1% THBY-2 mg of BSA/ml. Aliquots (15 μl) of bacteria were added to equal volumes of double dilutions of rhSLPI from 40 μM in phosphate buffer-THBY (or phosphate buffer-THBY only), in duplicate, giving final concentrations of rhSLPI from 20 μM, with BSA at 1 mg/ml and bacteria at 10⁵/ml, and incubated for 2 h at 37°C. Bacteria were then plated, and percent survival was calculated as described below. This experiment was performed twice in duplicate under the above conditions exactly and on more than six other occasions under a variety of slightly different conditions, all of which gave the same result.

(ii) Determination of the effect of SIC and rhSLPI on M1 GAS.M1 GAS were grown and prepared as described above and resuspended to a concentration of 2.5 × 10⁵/ml in phosphate buffer-1% THBY. Doubling dilutions of SIC from 20 μM were preincubated with 20 μM rhSLPI (both in phosphate buffer-1% THBY as described above) in a total volume of 15 μl (in duplicate) for 2 h at 37°C. Twelve microliters of M1 GAS at 2.5 × 10⁵/ml was added together with BSA to 1 mg/ml and incubated for a further 2 h at 37°C. Final overall concentrations were from 10 μM for SIC, 10 μM for rhSLPI, and 10⁵/ml for bacteria. Controls were bacteria in phosphate buffer-1% THBY, with dilutions of SIC from 10 μM or with 10 μM rhSLPI alone and with all suspensions containing BSA at 1 mg/ml. This experiment was performed twice in duplicate under the above conditions exactly and on more than six other occasions under a variety of slightly different conditions, all of which gave the same result.

(iii) Determination of the effect of HEL and SIC on S. suis.Overnight cultures of S. suis were grown at 37°C in THB, prepared as described above, and resuspended to a concentration of 2.5 × 10⁹/ml in phosphate buffer-1% THB. Doubling dilutions of SIC from 17.5 μM were combined with 17.5 μM HEL (both in phosphate buffer-THB) in a total volume of 15 μl and incubated overnight at 4°C. Twelve microliters of S. suis at 2.5 × 10⁹/ml was added together with BSA to 1 mg/ml, and the mixture was incubated for a further 15 min at 37°C. Final overall concentrations were from 8.5 μM for SIC, 8.5 μM for HEL, and 10⁹/ml for bacteria. Controls were bacteria in phosphate buffer-1% THB, dilutions of SIC from 8.5 μM, or 8.5 μM HEL alone, all of which contained BSA at 1 mg/ml.

In all antibacterial assays, 10-fold dilutions of the reaction mixtures were made in 100-μl volumes of phosphate buffer-THBY or -THB, plated on selective HBA plates, and incubated overnight at 37°C. The numbers of colonies were counted, and the results were plotted as percentages of bacteria that survived relative to the number of bacteria incubated in buffer only that survived ± the standard errors of the means (SEM). This experiment was performed twice in duplicate under the above conditions exactly and on more than four other occasions under a variety of slightly different conditions, all of which gave similar results.

HNE inhibition assays. (i) Titration of HNE activity and its inhibition by rhSLPI.Double dilutions of human neutrophil elastase (HNE) (CN Biosciences UK Ltd.) from 500 nM were made up in 0.1 M Tris-0.5 M NaCl, pH 7.5 (assay buffer). Eighteen microliters of each dilution was added to 100 μl of substrate solution [0.2 mM N-(methoxysuccinyl)-Ala-Ala-Pro-Val 4-nitroanilide; Sigma-Aldrich Co. Ltd.] in a flat-bottomed microtiter plate (Greiner Bio-One Ltd.) and incubated at room temperature for up to 1 h, and successive readings of optical density at 405 nm (OD₄₀₅) were taken. The optimum concentration of HNE for use in this system was 250 nM (7.37 μg/ml). Therefore, 10 μl of HNE at 14.75 μg/ml (500 nM) was added to 10-μl aliquots of double dilutions of rhSLPI from 11.7 μg/ml (1 μM), both of which were in Tris-NaCl assay buffer (experiment performed in duplicate), and incubated at 37°C for 15 min (final concentrations of 250 nM HNE and SLPI from 500 nM). Elastase activity was then measured as described above. A concentration of 500 nM SLPI was required to give complete inhibition of substrate digestion.

(ii) Effect of SIC on the inhibition of HNE activity by rhSLPI.Twenty microliters of SIC at 2.2 mg/ml (71 μM) was incubated with an equal volume of SLPI at 17.55 μg/ml (1.5 μM) or 20 μl of SLPI was incubated with an equal volume of HNE at 22.2 μg/ml (750 nM) for 15 min at 37°C. Twenty microliters of HNE or assay buffer was then added to the SIC and SLPI or 20 μl of SIC was added to the SLPI and HNE and incubated for a further 15 min at 37°C. Controls were mixtures of SLPI, HNE, and BSA at 2.5 mg/ml; SIC and HNE; HNE and BSA; SIC alone; SLPI and BSA; BSA alone; and buffer alone. All reaction mixtures were made up in duplicate. BSA was included in all the reaction mixtures not containing SIC as we found that merely having extra protein in the system greatly enhanced the function of HNE, and the substitution of BSA avoided this problem. Triplicate 18-μl aliquots of the duplicate reaction mixtures were then transferred to a microtiter plate, 100 μl of substrate solution was added, and the OD₄₀₅ was monitored. This experiment was performed four times in triplicate.

In a similar experiment, 6-μl aliquots of SIC at 350 μg/ml (11.3 μM) were combined with equal volumes of SLPI at 133 μg/ml (11.3 μM) or 6 μl of SLPI was combined with an equal volume of HNE at 3.33 μg/ml (0.113 μM) and incubated for 30 min at 37°C. Six microliters of HNE or buffer was then added to the SIC and SLPI mixture or 6 μl of SIC was added to the SLPI and HNE mixture, and the mixtures were incubated for a further 15 min at 37°C. Controls were a mixture of SLPI and HNE, a mixture of SIC and HNE, and SIC, SLPI, or HNE alone. Reducing Laemmli loading buffer (18 μl) was added, and the samples were boiled for 3 min, run on an SDS-12.5% PAGE gel, and stained with Coomassie blue.

Effect of SIC on the catalytic activity of lysozyme.Sequential dilutions of 3 parts SIC (from 0.5 mg/ml, 16.1 μM) and 1 part diluent were combined with HEL or human lysozyme at a constant 0.25 mg/ml (17.5 or 14.7 μM, respectively) in 15-μl volumes (both in 50 mM sodium phosphate buffer, pH 7) in duplicate and incubated overnight at 4°C. Sequential dilutions of HEL or human lysozyme alone from 0.5 mg/ml (3 parts lysozyme to 1 part diluent) were also made up and incubated as described above to provide a standard curve. Five-microliter portions of each set of reaction mixtures together with a set of standard-curve dilutions were added to 3-mm-diameter wells of duplicate agarose lysoplates containing Micrococcus lysodeikticus cell walls (catalog no. M3770; Sigma-Aldrich Co. Ltd.) at 0.5 mg/ml, according to the method described previously (7). The plates were incubated at room temperature for approximately 4 h, and the diameters of the zones of bacterial cell wall clearance were measured with a magnifying graticule with divisions of 0.1 mm. To confirm that SIC itself did not digest M. lysodeikticus cell walls, a comparable series of dilutions of SIC were investigated and no digestion was observed. The experiment with HEL was performed numerous times in duplicate, and that with human lysozyme was performed three times in duplicate. In another experiment to investigate the effects of temperature on the system, dilutions of SIC from 16.1 μM (3 parts SIC to 1 part diluent) were combined with 17.5 μM HEL, incubated overnight at 4 or 37°C, as were standard-curve dilutions, and assayed as described above. This experiment was performed three times in duplicate.

Effect of SLPI on the interaction between SIC and lysozyme.In order to ascertain whether there was any competition between SLPI and lysozyme for binding sites on SIC, double dilutions of SLPI from 28.5 μM were combined with 5 μM SIC and 10 μM HEL in 50 mM sodium phosphate buffer (in duplicate) in 15-μl volumes and incubated overnight at 4°C. Controls were 10 μM HEL, 5 μM SIC, 10 μM HEL plus 5 μM SIC, and buffer only, together with dilutions of HEL for a standard curve as described above. Samples (5 μl) of each reaction mix were added to duplicate lysoplates as described above, and zones of clearance were measured. This experiment was performed four times in duplicate. In order to confirm that SLPI itself did not digest M. lysodeikticus cell walls, a comparable series of dilutions of SLPI were investigated and no digestion was observed.

In all the above-described lysoplate assays, results were expressed as percentages of lysozyme activity remaining relative to the level of activity of the control (lysozyme alone).

Precipitation of HEL by SIC.Initial inhibition studies showed that when SIC and HEL were combined, a visible precipitate formed almost instantaneously. In order to investigate this further, double dilutions of SIC from 2 mg/ml (64 μM) were combined with HEL at 0.5 mg/ml (35 μM) in 100-μl volumes (in duplicate) and incubated overnight at 4°C. The precipitate was spun down and assayed by the method of Lowry et al. (19). Standard curves were constructed for SIC and HEL separately, and the mean of these curves was plotted to approximately represent a pellet comprising a mixture of the two proteins. This experiment was performed three times in duplicate.

Affinity purification of SIC on a lysozyme column.HEL was coupled to cyanogen bromide-activated Sepharose CL4B (14) at 8 mg/ml, and an 8-ml column was equilibrated in 50 mM Tris-10 mM EDTA, pH 7.5 (TE). One liter of M1 GAS overnight-culture supernatant was precipitated with 30% saturated ammonium sulfate at room temperature, adjusted to pH 5.5, resuspended in 30 ml of TE, dialyzed, and loaded onto the column at 4°C. The column was washed with TE, and the SIC eluted with a 20-column-volume steep sodium chloride gradient from 0 to 2 M.

SIC binds to rhSLPI, human lysozume, and HEL but not to lactoferrin as determined by ELISA.SIC bound strongly to plates coated with rhSLPI (Fig. 1a). Similarly, rhSLPI bound strongly to plates coated with SIC (data not shown). SIC also bound to plates coated with HEL (Fig. 1b) but only weakly to plates coated with human lysozyme (data not shown). However, human lysozyme bound to plates coated with SIC (Fig. 1c). No binding was observed between SIC and lactoferrin in either ELISA orientation.

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FIG. 1.

Binding of SIC to secretory leukocyte proteinase inhibitor, HEL, and human lysozyme by ELISA. In each panel, bar 1 is coating protein plus ligand plus first and second antibodies; bar 2 is coating protein plus first and second antibodies; bar 3 is coating protein plus second antibody; and bar 4 is coating protein plus ligand plus second antibody. Results are expressed as net ODs after deduction of background values from matching uncoated wells ± SEM. Where no error bars are shown, the SEM is too small to be seen. (a) Coating protein, secretory leukocyte proteinase inhibitor; ligand, SIC; (b) coating protein, HEL; ligand, SIC; (c) coating protein, SIC; ligand, human lysozyme.

Under physiological conditions SIC binds to rhSLPI at higher affinity than to HEL and not at all to lactoferrin.Equilibrium binding studies were performed using a hyperbolic-curve fit in order to characterize further the interactions of SIC with SLPI and HEL, and the thermodynamic properties were calculated. The results are shown in Tables 1 and 2. No binding was observed between SIC and lactoferrin. At 37°C the affinity of SLPI is substantially higher than that of lysozyme, with K_ds of 19.7 and 85.4, respectively, while at 4°C they are much the same. This is because the SIC-SLPI interaction shows little temperature dependence while the SIC-lysozyme interaction is highly temperature dependent. Approximately two molecules of SLPI bind to SIC, while approximately four molecules of lysozyme bind to SIC. The SLPI-SIC interaction shows a small positive enthalpy of 6.4 kJ/mol, while the lysozyme-SIC interaction shows a negative enthalpy of −34.6 kJ/mol. Similarly, the SLPI-SIC interaction shows a positive entropy of 0.111 kJ/mol/K while the lysozyme-SIC interaction shows a negative entropy of −0.035 kJ/mol/K.

SLPI kills the M1 strain of GAS and is inhibited by SIC.In order to investigate whether the M1 strain of GAS was susceptible to killing by SLPI, doubling dilutions of rhSLPI from 20 μM were incubated with the bacteria at 10⁵/ml. The results show that rhSLPI kills M1 GAS at concentrations similar to those published for other organisms and that virtually complete killing of the bacteria occurred at about 20 μM rhSLPI (Fig. 2a).

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FIG. 2.

SLPI kills the M1 strain of GAS, and killing is inhibited by SIC. (a) Doubling dilutions of SLPI from 20 μM were combined with bacteria at 10⁵/ml together with BSA at 1 mg/ml and incubated for 2 h at 37°C. Tenfold dilutions of the bacteria were plated. Results are from two experiments performed in duplicate. (b) Doubling dilutions of SIC from 40 μM were incubated with 40 μM SLPI for 2 h at 37°C. Bacteria at 2.5 × 10⁵/ml were added together with BSA to 1 mg/ml, incubated for a further 2 h at 37°C, and processed as described above. Final concentrations were 10⁵/ml for bacteria, from 10 μM for SIC, and 10 μM for SLPI. Controls were bacteria in 10 μM SLPI, buffer, or dilutions of SIC. Results are from a typical experiment performed in duplicate. In both panels, results are expressed as percentages of survival relative to the survival of bacteria incubated in buffer only ± SEM.

In order to investigate the effects of SIC upon the antibacterial properties of rhSLPI, double dilutions of SIC from 10 μM were combined with 10 μM rhSLPI and then incubated with M1 GAS at 10⁵/ml. SIC totally inhibited the killing of M1 GAS by 10 μM rhSLPI at an optimum concentration of 5 μM SIC, i.e., at a SIC/SLPI molar ratio of 1:2 (Fig. 2b). BSA was added throughout, as initial experiments showed that the addition of SIC alone caused enhanced growth compared to that of bacteria in phosphate buffer alone. This was shown to be an effect of protein concentration in the reaction mixture, the same effect being produced by the addition of comparable amounts of BSA, human serum albumin, or gelatin. The addition of BSA at 1 mg/ml throughout eliminated the problem.

SIC has no effect on HNE inhibition by rhSLPI.As the two functionally distinct domains of SLPI are structurally very similar, the potential effect of SIC upon elastase inhibition by rhSLPI was also investigated. Results showed that although SIC inhibited the antibacterial properties of SLPI as described above, it had no effect on the inhibition of elastase by SLPI. Figure 3a shows that preincubation of SLPI with SIC followed by the addition of HNE, or preincubation of SLPI with HNE followed by the addition of SIC, had no effect on the ability of SLPI to inhibit HNE activity. The second experiment, in which the reaction mixtures were analyzed by SDS-PAGE, essentially made the same point but also demonstrated that HNE digests SIC in the absence of SLPI; no digestion of SIC by HNE was observed in the presence of SLPI, irrespective of the order in which the reagents were combined (Fig. 3b). These experiments also showed that there is no competition for binding sites on SLPI between SIC and HNE.

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FIG. 3.

SIC has no effect on HNE inhibition by SLPI. (a) Twenty-microliter aliquots of 71 μM SIC were incubated with equal volumes of 1.5 μM SLPI or 20 μl of SLPI was incubated with an equal volume of 750 nM HNE for 15 min at 37°C. Twenty microliters of HNE or assay buffer was then added to the SIC and SLPI, and 20 μl of SIC was added to the SLPI and HNE; both mixtures were incubated for a further 15 min at 37°C. Triplicate 18-μl aliquots of duplicate reaction mixtures were transferred to a microtiter plate, 100 μl of the substrate solution was added, and the OD₄₀₅ was monitored. Bar 1, SIC plus SLPI; bar 2, SIC plus SLPI and then HNE; bar 3, SLPI plus HNE and then SIC; bar 4, SLPI plus HNE; bar 5, SIC plus HNE; bar 6, HNE plus BSA; bar 7, SIC; bar 8, SLPI plus BSA; bar 9, BSA; bar 10, buffer. Results are the means of results of three experiments performed in duplicate ± SEM. Error bars are too small to be seen. (b) Six-microliter aliquots of SIC and SLPI or of SLPI and HNE were incubated together for 30 min at 37°C. Six microliters of HNE or buffer was then added to the SIC and SLPI or 6 μl of SIC was added to the SLPI and HNE and incubated a further 15 min, as were controls. The samples were then run on an SDS-12.5% PAGE gel and stained with Coomassie blue. Molecular mass markers appear at both sides. Lane 1, SIC plus SLPI; lane 2, SIC plus SLPI and then HNE; lane 3, SLPI plus HNE and then SIC; lane 4, SLPI plus HNE; lane 5, SIC plus HNE; lane 6, SIC; lane 7, SLPI; lane 8, HNE (the quantity of HNE loaded was too small to be shown by Coomassie blue staining).

SIC inhibits killing of S. suis by HEL.In order to investigate the effects of SIC upon lysozyme function, SIC at different concentrations was preincubated with HEL and added to S. suis at 10⁹/ml. SIC partially inhibited killing of S. suis by HEL at an optimum SIC/HEL molar ratio of between 1:2 and 1:3 (Fig. 4). Even at the optimum molar ratio, only about 10% of the bacteria survived.

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FIG. 4.

Inhibition of lysozyme killing of S. suis by SIC. Double dilutions of SIC from 17 μM were incubated with 17 μM HEL overnight at 4°C. Equal volumes of S. suis at 2.5 × 10⁹ bacteria/ml were added together with BSA to 1 mg/ml and incubated for 15 min at 37°C. Final concentrations were 10⁹/ml for bacteria, from 8.5 μM for SIC, and 8.5 μM for HEL. Controls were bacteria in 8.5 μM HEL, dilutions of SIC, or buffer. Tenfold dilutions of the bacteria were plated. Results are expressed as percentages of surviving bacteria relative to the survival of bacteria incubated in buffer only ± SEM.

SIC inhibits the catalytic activity of lysozyme.Investigation of the catalytic activity of human lysozyme and HEL in the presence of SIC measured by digestion of bacterial cell walls showed that the maximum inhibition of HEL activity occurred at a SIC/HEL molar ratio of about 1:3 after overnight incubation at 4°C. With less SIC, the amount of inhibition diminished very sharply. The highest level of inhibition obtained was approximately 75%. Addition of more SIC does not increase the level of inhibition. A similar inhibition curve was obtained with human lysozyme, where the maximum inhibition of activity also occurred at a SIC/lysozyme molar ratio of approximately 1:3. The highest level of inhibition seen was never more than about 50% (Fig. 5a). However, after overnight incubation at 37°C, no inhibition of HEL activity was observed (Fig. 5b).

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FIG. 5.

Inhibition of the catalytic activities of human lysozyme and HEL by SIC. (a) Three in four dilutions of SIC from 16.1 μM were combined with 14.7 μM human lysozyme or 17.5 μM HEL and incubated overnight at 4°C. (b) Three in four dilutions of SIC from 16.1 μM were combined with 17.5 μM HEL and incubated overnight at 4 or 37°C. Residual lysozyme activity was assayed by the lysoplate method. Data are from three experiments performed in duplicate and are expressed as percentages of lysozyme activity remaining relative to the activity of the control (lysozyme alone) ± SEM.

SLPI competes with lysozyme for binding sites on SIC.In order to investigate whether there was competition between SLPI and HEL for binding to SIC, dilutions of SLPI were added to a mixture of SIC and HEL and the level of inhibition of HEL activity was assayed on lysoplates. SLPI at the highest concentration used completely negated the inhibition of HEL by SIC and restored HEL activity to 100%. SLPI competed with HEL for binding to SIC in a dose-dependent manner. No digestion was caused by SLPI alone. The results are shown in Fig. 6.

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FIG. 6.

Competition between SLPI and HEL for binding to SIC. Double dilutions of SLPI from 28.5 μM were incubated with 10 μM HEL and 5 μM SIC. HEL activity released (and hence the degree of competition by SLPI for binding to SIC) was assayed by the lysoplate method. Results are the results of four experiments performed in duplicate and are expressed as percentages of lysozyme activity relative to the level of activity of the control (10 μM lysozyme alone) ± SEM.

SIC does not break down SLPI or lysozyme.Incubation of SIC with SLPI, human lysozyme, or HEL does not result in the breakdown of any of the proteins. Coomassie blue-stained SDS-PAGE gels of the three protein combinations, and of individual proteins postincubation, showed no breakdown products (Fig. 7). It should be noted that SIC runs with an apparent molecular mass of around 45 kDa, although its actual molecular mass is 31 kDa, and that the slightly lower-molecular-mass band in the SIC tracks is a spontaneous breakdown product in which 33 amino acids have been lost from the N terminus (11).

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FIG. 7.

SIC does not break down SLPI, human lysozyme, or HEL. Five micrograms of SIC was incubated overnight at 4°C with 5 μg of human lysozyme, HEL, or SLPI or 5 μg of the individual proteins alone as controls. The samples were run on an SDS-12.5% PAGE gel and stained with Coomassie blue. Molecular mass markers are at both sides. Tracks: 1, SIC; 2, SIC plus SLPI; 3, SLPI; 4, SIC plus human lysozyme; 5, human lysozyme; 6, SIC plus HEL; 7, HEL; 8, SIC.

SIC precipitates HEL at 4°C.SIC precipitates HEL from solution at 4°C at an optimum HEL/SIC molar ratio of between 2.2 and 4.4:1, at which ratios greater than 80% of the total protein in the system is precipitated. The results are shown in Table 3.

Affinity purification of SIC on a lysozyme column.SIC protein from bacterial culture supernatant bound to HEL linked to a column of Sepharose CL4B and eluted at approximately 0.7 M sodium chloride in a single peak, as shown by SDS-PAGE. A typical elution profile is shown in Fig. 8. The eluted SIC was of a purity similar to that eluted from the HIC column as described in Materials and Methods. This HEL-Sepharose affinity procedure is now used as a final purification step after SIC is separated on the HIC column. SIC-containing fractions are collected from the HIC column, pooled, and dialyzed into TE as described above, run on an 8-ml lysozyme-Sepharose column, and eluted with a shallow 15-column-volume sodium chloride gradient from 0 to 1 M. SIC then elutes at around 0.3 M sodium chloride. SIC-containing fractions are then pooled and precipitated as described above.

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FIG. 8.

Affinity purification of SIC on a lysozyme-Sepharose column. HEL was coupled to cyanogen bromide-activated Sepharose CL4B to make an 8-ml affinity column and equilibrated with TE (pH 7.5). A 30% ammonium sulfate precipitate from a 1-liter M1 GAS overnight-culture supernatant (resuspended and dialyzed into TE) was applied to the column and eluted in a 20-column-volume sodium chloride gradient from 0 to 2 M. SIC eluted at around 0.3 M sodium chloride.