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Infection and Immunity, January 2008, p. 179-188, Vol. 76, No. 1
0019-9567/08/$08.00+0     doi:10.1128/IAI.00837-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

L-Ficolin/Mannose-Binding Lectin-Associated Serine Protease Complexes Bind to Group B Streptococci Primarily through N-Acetylneuraminic Acid of Capsular Polysaccharide and Activate the Complement Pathway{triangledown}

Youko Aoyagi,1 Elisabeth E. Adderson,2 Craig E. Rubens,3 John F. Bohnsack,4 Jin G. Min,5 Misao Matsushita,5 Teizo Fujita,6 Yoshiyuki Okuwaki,1 and Shinji Takahashi1*

Division of Microbiology, Joshi-Eiyoh (Kagawa Nutrition) University, Sakado, Japan,1 Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee,2 Department of Pediatrics, Children's Hospital and Regional Medical Center, University of Washington, Seattle, Washington,3 Department of Pediatrics, University of Utah Health Sciences Center, Salt Lake City, Utah,4 Department of Applied Biochemistry, Tokai University, Hiratsuka, Japan,5 Department of Immunology, Fukushima Medical University, Fukushima, Japan6

Received 18 June 2007/ Returned for modification 31 July 2007/ Accepted 8 October 2007


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ABSTRACT
 
Group B streptococci (GBS) are the most common cause of neonatal sepsis and meningitis. Most infants who are colonized with GBS at birth do not develop invasive disease, although many of these uninfected infants lack protective levels of capsular polysaccharide (CPS)-specific antibody. The lectin pathway of complement is a potential mechanism for initiating opsonization of GBS with CPS-specific antibody-deficient serum. In this study, we determined whether mannose-binding lectin (MBL)/MBL-associated serine protease (MASP) complexes and L-ficolin/MASP complexes bind to different strains of GBS to activate the lectin pathway, and we identified the molecules recognized by lectins on the GBS surface. We found that MBL did not bind to any GBS examined, whereas L-ficolin bound to GBS cells of many serotypes. L-ficolin binding to GBS cells correlated with the CPS content in serotypes Ib, III (restriction digestion pattern types III-2 and III-3), and V but not with the group B-specific polysaccharide (GBPS) content or with the lipoteichoic acid (LTA) content. L-ficolin bound to purified CPS and GBPS in a concentration-dependent manner but not to purified LTA. All strains to which L-ficolin/MASP complexes bound consumed C4. When N-acetylneuraminic acid (NeuNAc) was selectively removed from GBS cells by treatment with neuraminidase, the reduction in L-ficolin binding was correlated with the amount of NeuNAc removed. Additionally, L-ficolin was able to bind to wild-type strains but was able to bind only weakly to unencapsulated mutants and a mutant strain in which the CPS lacks NeuNAc. We concluded that L-ficolin/MASP complexes bind to GBS primarily through an interaction with NeuNAc of CPS.


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INTRODUCTION
 
Group B streptococci (GBS) (Streptococcus agalactiae) are the most common cause of neonatal sepsis and meningitis (4). GBS are classified into nine serotypes (serotypes Ia, Ib, and II through VIII) based on the immunologic reactivity of the capsular polysaccharide (CPS). The serotypes can be subclassified by numerical analysis of restriction digest patterns (RDPs) of chromosomal DNA (35), and the CPS content tends to be different in different RDP types of each serotype (42).

Maternal CPS-specific antibodies (Ab) are transferred to the fetus via the placenta and protect colonized neonates from invasive GBS disease. Most infants who are colonized with GBS at birth do not develop invasive disease, although many of these uninfected infants lack protective levels of CPS-specific Ab (27, 28). Indeed, although the amount of serotype III CPS-specific immunoglobulin G (IgG) correlates with the efficiency of opsonophagocytic killing by polymorphonuclear leukocytes in vitro, serum deficient in CPS-specific IgG still mediates significant opsonophagocytic killing in the presence of both Ca2+ and Mg2+ by a process that involves activation of the complement pathway (8-10). Similarly, the complement-derived chemoattractant C5a is produced when GBS are incubated with CPS-specific IgG-depleted serum containing Ca2+ and Mg2+ (44).

The lectin pathway of the complement system is a potential mechanism for initiating opsonization of GBS with CPS-specific Ab-deficient serum because the C3 convertase C4b2a complexes of the lectin pathway are generated by a mechanism that is both Ca2+ dependent and Ab and C1 independent. Three different carbohydrate recognition components of the lectin pathway have been described: mannose-binding lectin (MBL), L-ficolin, and H-ficolin (11). All recognition components consist of homotrimers of a single polypeptide chain with an N-terminal collagen-like domain, a neck region, and a C-terminal carbohydrate recognition region and are present as higher-order oligomers of the homotrimeric subunits. In serum, the oligomers form complexes with MBL-associated serine proteases (MASP) (namely, MASP-1, MASP-2, and MASP-3) to form a lectin pathway activation complex (41). One of these MASP, MASP-2, cleaves C4 and C2 to generate the C4b2a complex (40). Although it has been shown that L-ficolin binds to serotype III GBS CPS (3) and lipoteichoic acid (LTA) (29) and that MBL has low affinity for GBS (36, 45), the studies were performed with restricted numbers of GBS strains. The ligand for both L-ficolin and MBL, N-acetylglucosamine (GlcNAc), is one of five monosaccharides incorporated into the CPS of GBS belonging to all serotypes (17-19, 21, 49-51) except serotype VI CPS (46) and serotype VIII CPS (20) and is a constituent of the group B-specific polysaccharide (GBPS) (33). In the present study, we determined whether L-ficolin/MASP complexes and MBL/MASP complexes bind to various RDP types of GBS to activate the lectin pathway of complement, and we identified the molecules recognized by lectins on the GBS surface.


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MATERIALS AND METHODS
 
Bacterial strains and capsule-deficient isogenic mutant. GBS isolates (56 strains) whose RDP types had been identified previously (43) were used. CPS-deficient and N-acetylneuraminic acid (NeuNAc)-deficient isogenic mutants were used to examine the contributions of CPS and NeuNAc to L-ficolin binding (Table 1). Bacteria were cultured overnight in Todd-Hewitt broth (THB) (BBL, Microbiology Systems, Cockeysville, MD) and then inoculated at a 1:20 dilution into fresh THB and incubated at 37°C for 90 to 120 min. Bacteria were washed twice with 10 mM Tris-HCl-150 mM NaCl (TBS) (pH 7.4) containing 0.02% NaN3 and resuspended in the same buffer to an optical density at 600 nm (OD600) of 0.60. Each 1 ml of suspension at an OD600 of 0.60 contained 168 µg (dry weight) of cells and between 0.4 x 108 and 1.8 x 108 CFU (44). Bacteria used for neuraminidase treatment were washed twice with 20 mM phosphate buffer-150 mM NaCl-0.02% NaN3 (pH 5.1) and resuspended in the same buffer to an OD600 of 0.60. Each bacterial suspension (2 ml) was incubated with or without 40 U neuraminidase type V (Sigma-Aldrich, St. Louis, MO) overnight at 37°C. Bacteria were washed twice and resuspended in TBS containing 0.02% NaN3 to an OD600 of 0.60.


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  		TABLE 1. Bacterial strains

L-ficolin/MASP complex purification. L-ficolin/MASP complexes were purified from normal human serum by affinity chromatography on GlcNAc-agarose (Sigma-Aldrich), followed by affinity chromatography on asialofetuin-Sepharose made by conjugating asialofetuin (Sigma-Aldrich) to CNBr-activated Sepharose 4B (GE Healthcare, Buckinghamshire, United Kingdom) as previously described (6, 31). Purified material was successively passed through affinity columns made by conjugating anti-human IgG and anti-human IgM monoclonal antibodies (MAbs) (Zymed Laboratories, San Francisco, CA) to HiTrap N-hydroxy succinimide-activated high-performance columns (HiTrap NHS-activated HP; GE Healthcare). The concentration of the complexes was determined using a Lowry assay kit (Sigma-Aldrich). Affinity-purified L-ficolin/MASP complexes prepared in this manner have previously been shown to be composed of L-ficolin, MASP-1, -2, and -3, and a small MBL-associated protein (6).

Purification of GBS serotype-specific CPS and GBPS. CPS were extracted from GBS strains i45 (serotype Ia), 590448 (serotype Ib), 874391 (serotype III), Washington (serotype II), i16 (serotype V), 853009 (serotype VI), and 872808 (serotype VIII). Supernatants of late-exponential-phase bacteria grown in THB were filtered and concentrated with an ultrafilter and ethanol precipitation as previously described (3). The crude CPS material was treated with 1 M NaOH overnight at 37°C to depolymerize GBPS and LTA, and the preparation was then purified by gel filtration with Sephacryl S-300 HR (GE Healthcare), followed by ion-exchange chromatography with DEAE-Sepharose CL-6B (GE Healthcare) as previously described (17). The purified CPS was dialyzed in water and lyophilized. CPS was purified in the same manner except that the NaOH treatment was omitted. GBPS was extracted from the CPS-deficient isogenic mutant 874391/cps– by using the same method that was used for CPS extraction except that the NaOH treatment was omitted. Type and group B antigenic activities in preparations were assayed by the method of Ouchterlony (37) with rabbit antiserum against the type-specific CPS provided by F. Gondaira (Denka Seiken, Tokyo, Japan) and by a latex agglutination procedure (B Strepto AD; Denka Seiken), respectively. The NeuNAc (2) content of the purified CPS and the GlcNAc (13) content of the purified GBPS were confirmed by chemical analysis as previously described. The LTA concentrations in the purified CPS and GBPS preparations were less than 10 pmol/1,000 pmol NeuNAc and less than 10 pmol/1,000 pmol GlcNAc, as measured by an enzyme-linked immunosorbent assay (ELISA) as described below.

LTA purification. LTA were extracted from GBS strains i45 (serotype Ia), 590448 (serotype Ib), 874391 (serotype III), Washington (serotype II), i16 (serotype V), 853009 (serotype VI), 872808 (serotype VIII), and 874391/cps– as previously described (34). Briefly, a pellet of late-exponential-phase bacteria grown in THB was mixed with an equal volume of n-butanol while it was being stirred for 30 min at room temperature. After centrifugation at 10,000 x g for 20 min, the aqueous phase was lyophilized and resuspended in 15% n-propanol in 100 mM ammonium acetate-acetic acid buffer (pH 4.7). LTA was purified by hydrophobic interaction chromatography on octyl-Sepharose (GE Healthcare). LTA obtained in this way was concentrated on a YM1 ultrafilter (Millipore, Massachusetts) and resolved in 5 mM ammonium acetate-acetic acid buffer (pH 4.7). The antigenic activity of LTA was assayed by an ELISA as described below. There were no differences in LTA antigenic activity among LTA preparations. The phosphorus (1) and GlcNAc (13) contents of the purified LTA were confirmed by chemical analysis as previously described. The GlcNAc concentration in every purified LTA preparation was less than 1 nmol/100 nmol phosphorus. The CPS concentration (expressed as the NeuNAc concentration) in every purified LTA preparation was less than 1 nmol/100 nmol phosphorus.

NeuNAc content of CPS. Bacteria (1,000 µl of a suspension having an OD600 of 0.6 in TBS containing 0.02% NaN3) were washed with water and resuspended in 0.1 M HCl. The NeuNAc was extracted from the bacterial suspension by hydrolysis at 84°C for 20 min, and the NeuNAc content of the extract was determined by chemical analysis as previously described (2).

GBPS content of bacteria. The GBPS content of bacteria was estimated using a latex agglutination procedure. Bacteria (1,000 µl of a suspension having an OD600 of 0.6 in TBS containing 0.02% NaN3) were pelleted, resuspended in 500 µl of 20 mM phosphate buffer (pH 6.8)-10 mM MgCl2-40% (wt/vol) sucrose-0.02% NaN3-20 U/ml mutanolysin (Sigma-Aldrich), and incubated for 20 h at 37°C to digest the peptidoglycan of cell walls, and the protoplasts were removed by centrifugation. The cell wall extract (40 µl), diluted 200-fold with 20 mM phosphate buffer (pH 7.4)-150 mM NaCl, was added to the same volume of a twofold dilution of an anti-GBPS Ab-sensitized latex particle suspension (B Strepto AD; Denka Seiken) in wells of a microtiter plate which had an extremely low affinity for organic substances (Assay plate; Asahi Techno Glass, Tokyo, Japan). After mixing with a microtiter mixer for 30 s, the OD660 was measured immediately. Five twofold dilutions of the purified GBPS (125 to 2,000 pmol/ml GlcNAc) were prepared as standards, and the GBPS concentration of the cell wall extract was estimated from the standard curve. For all strains tested, GBPS was detected in the cell wall extract but not in the protoplast (less than 25 nmol GlcNAc per ml of a suspension having an OD600 of 0.6), indicating that there was efficient digestion of the cell walls.

LTA content of the bacterial cell surface. The LTA content of the bacterial cell surface was estimated using a modification of an ELISA as previously described (7). Briefly, bacteria (80 µl of a suspension having an OD600 of 0.12 in TBS containing 0.02% NaN3) were coated onto wells of microtiter plates (ELISA plates; Asahi Techno Glass) overnight at 4°C. Wells were blocked by incubation for 1 h at room temperature with 200 µl of a 1-µg/ml human serum albumin (Sigma-Aldrich) solution in TBS containing 0.05% Tween 20 (TBST). Anti-LTA MAb (clone 55; Hycult Biotechnology, Uden, Netherlands) in TBST, which recognized the polyglycerophosphate backbone of LTA, was added and incubated overnight at 4°C. After washing with TBST, 80 µl of a solution containing 1 µg/ml alkaline phosphatase-conjugated goat anti-mouse Ig antibody (Zymed Laboratories, San Francisco, CA) in TBST was added and incubated for 1 h at room temperature. After washing with TBST, LTA was detected by using the ELISA amplification system (Invitrogen, Carlsbad, CA). Dilutions of purified LTA (20 to 120 pmol phosphorus per well) extracted from strain 874391/cps– in TBS were prepared as standards, and the LTA concentration was estimated from the standard curve.

L-ficolin and MBL binding to bacterial cells. Normal human serum with known concentrations of L-ficolin/MASP complexes (3.9 µg/ml) and MBL/MASP complexes (1.4 µg/ml) was used in the assays. A binding mixture (300 µl) consisting of a bacterial suspension having an OD600 of 0.60 and 8% serum in 10 mM Tris-HCl-10 mM HEPES (Invitrogen)-150 mM NaCl-4 mM CaCl2-0.4% human serum albumin (Sigma-Aldrich)-0.05% Triton X-100-0.02% NaN3 (pH 7.4) (binding buffer) was incubated for 30 min at 37°C with shaking. In some experiments, four fourfold dilutions of bacteria (final OD600, 0.038, 0.15, 0.60, and 2.4) were prepared and incubated with serum in the same manner. After centrifugation, the L-ficolin or MBL remaining in the supernatants was quantified by ELISA. Serum (8%) without bacteria in binding buffer was treated in the same manner as preparations containing bacteria and used as a standard. The wells of microtiter plates were coated by incubation overnight at 4°C with 80 µl of a 1-µg/ml solution of anti-L-ficolin MAb (GN5; HyCult Biotechnology) or anti-MBL MAb (3E7) (32) in phosphate-buffered saline (PBS). The wells were blocked and washed as described above. The supernatant (80 µl) of the binding mixture was added to the wells, and the plates were incubated overnight at 4°C. After washing with TBS containing 0.05% Tween 20 and 2 mM CaCl2 (TBST/Ca), 80 µl of a solution containing 1 µg/ml of anti-L-ficolin MAb (2F5) (32) conjugated to alkaline phosphatase or 80 µl of a solution containing 1 µg/ml of anti-MBL MAb (Hyb 131-1; Abcam, Cambridge, United Kingdom) conjugated to alkaline phosphatase in TBST/Ca was added and incubated for 1 h at room temperature. Alkaline phosphatase conjugation of MAb was performed using an EZ-Link maleimide-activated alkaline phosphatase kit (Pierce Biotechnology, Rockford, IL). After washing with TBST/Ca, L-ficolin or MBL was detected by using the ELISA amplification system. Four twofold dilutions of the standard (12.5, 25, 50, and 100%) in binding buffer were prepared, the percentage of L-ficolin or MBL remaining in the supernatant was estimated from the standard curve, and the percentage of binding was calculated as follows: 100% – percentage remaining.

C4 consumption by L-ficolin/MASP complexes bound to bacteria. C4 activation by L-ficolin/MASP complexes bound to bacteria was quantified using a modification of a previously described C4 consumption assay (24). A binding mixture (1,000 µl) consisting of a bacterial suspension having an OD600 of 0.60 and 8% serum in 10 mM Tris-HCl-10 mM HEPES-500 mM NaCl-4 mM CaCl2-0.4% human serum albumin-0.05% Triton X-100-0.02% NaN3 (high-salt binding buffer) was incubated overnight at 4°C with rotation. Incubation at 4°C in the presence of 500 mM NaCl prevents activation of endogenous C4 and dissociates C1 complexes that are composed of C1q, C1r, and C1s (38). After centrifugation, the bacteria were washed twice with TBS containing 2 mM CaCl2 and 0.02% NaN3 and resuspended in 4 mM barbital-150 mM NaCl-2 mM CaCl2-0.05% Tween 20-0.02% NaN3 (pH 7.4) (activation buffer) to an OD600 of 0.60. An activating mixture (300 µl) consisting of a bacterial suspension having an OD600 of 0.60 and 1 µg/ml C4 (Sigma-Aldrich) in activation buffer was incubated for 30 min at 37°C with shaking. After centrifugation, the intact C4 remaining in the supernatants was quantified by using the assay described below. In some experiments, four fourfold dilutions of bacteria (final OD600, 0.038, 0.15, 0.60, and 2.4) were prepared from a bacterial suspension having an OD600 of 0.60 that had been incubated with 8% serum and were then incubated with C4 in the same manner. An activating mixture consisting of C4 and bacteria that had not been incubated with serum in binding buffer was treated in the same manner and used as a standard.

The intact C4 remaining in the supernatant was quantified using microtiter plates precoated with MBL/MASP complexes. The wells of microtiter plates were coated by incubation overnight at 4°C with 80 µl of a solution containing 1 µg/ml of mannan (Sigma-Aldrich) in PBS, blocked, and washed as described above. The wells were incubated overnight at 4°C with 80 µl of 8% serum in high-salt binding buffer and washed with TBST/Ca. The supernatant (80 µl) of the activating mixture was added to the wells, and the plates were incubated for 1 h at 37°C. After washing with TBST/Ca, 80 µl of a solution containing 1 µg/ml anti-C4 MAb (Hyb 162-02; Abcam) in TBST/Ca was added and incubated overnight at 4°C. After washing with TBST/Ca, 80 µl of a solution containing 1 µg/ml alkaline phosphatase-conjugated goat anti-mouse Ig Ab in TBST/Ca was added and incubated for 1 h at room temperature. After washing with TBST/Ca, C4b bound to the well was detected by using the ELISA amplification system. Dilutions of the standard (12.5, 25, 50, 75, and 100%) in activating buffer were prepared, the percentage of intact C4 remaining in the supernatant was estimated from the standard curve, and the percentage of consumption was calculated as follows: 100% – percentage of C4 remaining.

Inhibition of L-ficolin binding by purified CPS, GBPS, LTA, or monosaccharide. Inhibition of L-ficolin binding to bacteria or GlcNAc coated onto microtiter wells by purified soluble CPS, GBPS, LTA, or monosaccharide was quantified using a modification of a previously described inhibition assay (14). The wells of microtiter plates were coated by incubation overnight at 4°C with 80 µl of a bacterial suspension having an OD600 of 0.125 in TBS containing 0.02% NaN3 or with 80 µl of a solution containing 2.16 µg/ml of bovine serum albumin-conjugated GlcNAc (containing 1 µg/ml GlcNAc) (Dextra Laboratories, Reading, United Kingdom) in PBS, blocked, and washed as described above. Various concentrations of purified CPS, GBPS, or LTA (40 µl) or of six monosaccharides (NeuNAc, GlcNAc, glucose, galactose, rhamnose, and glucitol) (40 µl) that comprise CPS and GBPS in binding buffer were added to the well, followed by 40 µl of a solution containing 2 µg/ml of purified L-ficolin/MASP complexes in binding buffer, and incubated overnight at 4°C. After washing with TBST/Ca, 80 µl of a solution containing 1 µg/ml anti-L-ficolin MAb (GN5) in TBST/Ca was added and incubated for 1 h at room temperature. After washing with TBST/Ca, 80 µl of a solution containing 1 µg/ml alkaline phosphatase-conjugated goat anti-mouse Ig Ab in TBST/Ca was added and incubated for 1 h at room temperature. After washing with TBST/Ca, L-ficolin bound to bacteria or GlcNAc coated onto microtiter wells was detected by using the ELISA amplification system. Dilutions of a solution containing 2 µg/ml L-ficolin/MASP complexes (12.5, 25, 50, 75, and 100%) in activating buffer were prepared as standards and were treated in the same manner as samples except that inhibitors were not added. The percentage of L-ficolin bound to bacteria or GlcNAc was estimated from the standard curve as described above, and the concentration of an inhibitor that resulted in 50% inhibition (IC50) was determined from the inhibition curve.


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RESULTS
 
Binding of L-ficolin/MASP and MBL/MASP complexes to intact GBS cells and consumption of complement C4. The ability of L-ficolin and MBL to bind to GBS belonging to a variety of different serotypes and RDP types was examined by incubating intact cells with serum. The CPS content expressed as the NeuNAc content of bacterial cells, the GBPS content of bacterial cells, and the LTA content of the bacterial surface of the 56 GBS strains used are shown in Fig. 1. Strains to which less than 30% of the L-ficolin or MBL bound were tested to determine whether the binding was dependent on the bacterial concentration, and binding was designated nonspecific if no concentration-dependent binding was observed. The ability of L-ficolin to bind to intact bacteria varied according to the serotype and RDP type. L-ficolin bound significantly to all strains of serotypes Ib (five strains), V (12 strains), VI (five strains), and VIII (five strains) but not to any strain of serotype Ia (nine strains) or II (six strains). For serotype III strains, L-ficolin bound to all strains of RDP types III-2 (five strains) and III-3 (five strains) but not to strains of RDP type III-1 (two strains) or III-4 (two strains). L-ficolin binding increased with increasing CPS content in serotypes Ib, III (RDP types III-2 and III-3), and V, whereas L-ficolin binding was consistently high in serotypes VI and VIII (Fig. 2A). On the other hand, there was no correlation between L-ficolin binding and GBPS content or between L-ficolin binding and LTA content (Fig. 2B and 2C). MBL did not bind to any bacteria tested, a finding consistent with previous observations (36, 45). All strains to which L-ficolin/MASP complexes bound consumed C4 (Fig. 3A). C4 consumption appeared to be due to L-ficolin/MASP complexes since MBL did not bind to GBS, but there was no correlation between the amount of C4 consumption and the NeuNAc concentration; therefore, there was no correlation between the amount of C4 consumption and the amount of L-ficolin binding. This poor correlation did not appear to be due to experimental factors because C4 consumption was dependent on the bacterial concentration in individual strains when bacteria were incubated with serum to bind L-ficolin/MASP complexes, washed, diluted, and incubated with C4 (Fig. 3B). Because the bacteria were diluted after incubation with serum, the C4 consumption in these experiments was correlated with the amount of L-ficolin/MASP complexes present on the bacterial surface during the incubation with C4.


Figure 1
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  		FIG. 1. CPS content expressed as the NeuNAc content of bacterial cells and RDP types (A), GBPS content of bacterial cells and RDP types (B), and LTA content of the bacterial cell surface and RDP types (C). NeuNAc was extracted from bacteria, and the amount was determined by a chemical analysis. The GBPS content was estimated by a latex agglutination procedure using cell wall extract, and the concentration is expressed as the GlcNAc concentration. The LTA content was estimated by an ELISA using bacteria coated onto microtiter wells, and the concentration is expressed as the phosphorus concentration. RDP types were determined previously (43). The bars indicate the means for strains belonging to different RDP types. The error bars indicate the minimal and maximal concentrations for each RDP type.


Figure 2
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  		FIG. 2. Correlations between L-ficolin binding and the CPS content of bacteria (A), between L-ficolin binding and the GBPS content of bacteria (B), and between L-ficolin binding and the LTA content of bacteria (C). Bacteria were incubated with serum at 37°C for 30 min, and the amount of L-ficolin remaining in the supernatant was estimated. Concentrations are expressed as the NeuNAc concentration for CPS, as the GlcNAc concentration for GBPS, and as the phosphorus concentration for LTA. The data are the means of three experiments. The lines are correlation curves for the log CPS concentration and the percentage of L-ficolin binding for serotypes Ib (r2 = 0.748), III (RDP types III-2 and III-3) (r2 = 0.754), and V (r2 = 0.756).


Figure 3
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  		FIG. 3. (A) Correlation between C4 consumption of bacterium-bound L-ficolin/MASP complexes and CPS content of bacteria. Bacteria were incubated with serum overnight at 4°C in the presence of 500 mM NaCl, washed, and incubated with C4 at 37°C for 30 min. The amount of intact C4 remaining in the supernatant was estimated. The data are the means of three experiments. (B) C4 consumption of various concentrations of L-ficolin/MASP complex-bound bacteria. Strains 590448 (serotype Ib), 874391 (serotype III), i16 (serotype V), 853009 (serotype VI), and 872808 (serotype VIII) were incubated with serum overnight at 4°C in the presence of 500 mM NaCl to bind L-ficolin/MASP complexes, washed, diluted, and incubated with C4 at 37°C for 30 min. The amount of intact C4 remaining in the supernatant was estimated. The data are the means ± standard deviations of three experiments.

Inhibition of L-ficolin binding by purified CPS, GBPS, and LTA. The ability of L-ficolin to bind to purified soluble CPS (which had been treated with NaOH to avoid GBPS and LTA contamination), GBPS, and LTA was determined using an inhibition assay of L-ficolin binding to bacteria or GlcNAc coated onto microtiter wells. Purified CPS from serotype Ib, III, V, VI, and VIII bacteria inhibited L-ficolin binding to bacteria from which CPS was extracted in a concentration-dependent manner (Fig. 4A). Purified CPS from serotype Ia and II bacteria also inhibited L-ficolin binding to GlcNAc despite the observation that L-ficolin did not bind to serotype Ia and II bacteria (Fig. 4A). Purified GBPS from the 874391/cps– strain inhibited L-ficolin binding to serotype Ib, III, V, VI, and VIII bacteria coated onto microtiter wells in a concentration-dependent manner (Fig. 4B). The IC50 of GBPS (153.8 nmol/ml) for L-ficolin binding to serotype VIII bacteria was 4 to 13 times higher than the IC50 of GBPS for L-ficolin binding to serotype Ib, III, V, and VI bacteria (12.1 to 36.1 nmol/ml). Purified LTA from serotype Ib, III, V, VI, and VIII bacteria did not inhibit L-ficolin binding to each of the bacteria from which the LTA had been extracted. Purified LTA from serotype Ia and II bacteria also did not inhibit L-ficolin binding to GlcNAc coated onto microtiter wells at the equimolar concentrations of CPS and GBPS examined (Fig. 4C). In order to confirm the ability of L-ficolin to bind to LTA, 80 ng of purified LTA from strain 874391/cps–, which was not contaminated with CPS but was contaminated with a small amount of GBPS (less than 1%), was coated onto microtiter wells and reacted with 10 to 80 ng purified L-ficolin/MASP complex for 30 min at 37°C. Essentially no L-ficolin binding to LTA was observed compared to the binding to GlcNAc-coated microtiter wells (less than 4%) (data not shown).


Figure 4
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  		FIG. 4. Inhibitory effects of CPS (A), GBPS (B), and LTA (C) on L-ficolin binding to bacteria or GlcNAc. Various concentrations of purified CPS and LTA from strains 590448 (serotype Ib), 874391 (serotype III), i16 (serotype V), 853009 (serotype VI), and 872808 (serotype VIII) were incubated in microtiter wells coated with each bacterium from which CPS and LTA were extracted, and various concentrations of purified CPS and LTA from strains i45 (serotype Ia) and Washington (serotype II), whose L-ficolin did not bind to whole cells, were incubated in microtiter wells coated with GlcNAc in the presence of purified L-ficolin/MASP complexes. Various concentration of purified GBPS from 874391/cps– were incubated in microtiter wells coated with serotype Ib, III, V, VI, and VIII bacteria in the presence of purified L-ficolin/MASP complexes. The amounts of L-ficolin bound to bacteria and GlcNAc were estimated. Concentrations are expressed as the NeuNAc concentration for CPS, as the GlcNAc concentration for GBPS, and as the phosphorus concentration for LTA. The data are the means ± standard deviations of three experiments.

GBS CPS are known to modify NeuNAc residues by O acetylation at carbon position 7, 8, or 9, and NaOH treatment eliminates this modification (26). Purified CPS from the strains that had not been treated with NaOH and that were therefore contaminated with a small amount of GBPS and LTA had IC50s ranging from 36 to 59 nmol/ml for L-ficolin binding to GlcNAc coated onto microtiter wells (data not shown). These IC50s were almost identical to the IC50s (46 to 86 nmol/ml) of purified CPS which had been treated with NaOH for L-ficolin binding to GlcNAc coated onto microtiter wells (data not shown).

Inhibition of L-ficolin binding by monosaccharides comprising CPS and GBPS. Inhibition of L-ficolin binding to bacteria coated onto microtiter wells by the monosaccharides that comprise CPS and GBPS was measured to determine the ligand for L-ficolin on bacterial cells. The inhibitory abilities of NeuNAc and GlcNAc are shown in Fig. 5. Other monosaccharides, including glucose, galactose, rhamnose, and glucitol, at equimolar concentrations did not inhibit L-ficolin binding to serotype Ib, III, V, VI and VIII bacteria coated onto microtiter wells (data not shown). The IC50s of NeuNAc for L-ficolin binding to serotype Ib, III, V, VI, and VIII bacteria were almost identical (11.1 to 12.2 nmol/ml), and the IC50s of GlcNAc for serotype Ib, III, V, and VI bacteria were restricted to a narrow range (55.8 to 88.9 nmol/ml). On the other hand, GlcNAc did not inhibit L-ficolin binding to serotype VIII bacteria even at the highest concentration used (Fig. 5). Of interest, the IC50s of purified CPS (54 to 129.1 nmol/ml) from serotype Ib, III, V, VI, and VIII bacteria (Fig. 4A) were 86 to 226 times lower than those of NeuNAc (11.1 to 12.2 µmol/ml) (Fig. 5).


Figure 5
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  		FIG. 5. Inhibitory effects of NeuNAc and GlcNAc on L-ficolin binding to bacteria. Various concentrations of NeuNAc and GlcNAc were incubated in microtiter wells coated with bacterial strains 590448 (serotype Ib), 874391 (serotype III), i16 (serotype V), 853009 (serotype VI), and 872808 (serotype VIII) in the presence of purified L-ficolin/MASP complexes. The amounts of L-ficolin bound to bacteria were estimated. The data are the means ± standard deviations of three experiments.

Role of NeuNAc residues in CPS on intact bacterial cells as a ligand for L-ficolin. NeuNAc is a nonreducing terminal side residue in all serotype CPS. Bacterial cells from which NeuNAc was selectively removed by treatment with neuraminidase were used to define the role of NeuNAc in CPS on intact bacteria as a ligand for L-ficolin. The ability of neuraminidase to remove NeuNAc was different in different strains. The amount of residual NeuNAc on neuraminidase-treated cells of serotype VIII strain 872808 was high compared with the amounts on strains belonging to other serotypes (Fig. 6B). In serotype II and VIII CPS, NeuNAc is present as a monosaccharide side chain, while the other serotypes have disaccharide (serotypes III and VI) or trisaccharide (serotypes Ia, Ib, and V) side chains with NeuNAc as the terminal residue (17-21, 46, 49-51). NeuNAc was also not removed efficiently from a serotype II isolate by treatment with neuraminidase (91.7% of the NeuNAc remained on strain Washington [data not shown]). Presumably, L-ficolin was able to bind to the NeuNAc remaining on the cells after the treatment with neuraminidase, as L-ficolin bound to both the neuraminidase-treated bacteria and the intact bacteria (Fig. 6A). When the relative L-ficolin binding to neuraminidase-treated bacteria was calculated from the curve for binding of L-ficolin to intact bacteria as a standard for each serotype, the relative L-ficolin binding correlated with the relative amount of NeuNAc that remained (Fig. 6B), suggesting that NeuNAc is the major ligand for L-ficolin on the GBS cell surface. This conclusion was strengthened by additional experiments using isogenic mutants of serotype III strains. L-ficolin was able to bind to wild-type strains 874391 and COH1, but it bound only weakly to unencapsulated mutants 874391/cps– and COH1-13 (Fig. 7A and D) despite the fact the latter strains had the same levels of GBPS as their parental strains (Fig. 7B) and higher levels of LTA (Fig. 7C). Furthermore, L-ficolin bound only weakly to COH1-11, an isogenic mutant strain in which the CPS lacks NeuNAc, compared to the L-ficolin binding to COH-1, its parental strain (Fig. 7D). The CPS production by NeuNAc-deficient isogenic mutant COH1-11 is approximately 20% of the CPS production by parental strain COH1 (5). A lack of L-ficolin binding to COH1-11 was demonstrated even when high concentrations of bacteria were used to compensate for the 80% reduction in CPS production by COH1-11. These observations demonstrate that L-ficolin binds to CPS primarily through NeuNAc.


Figure 6
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  		FIG. 6. (A) L-ficolin binding to neuraminidase-treated and intact bacteria. Neuraminidase-treated and intact bacterial strains 590448 (serotype Ib), 874391 (serotype III), i16 (serotype V), 853009 (serotype VI), and 872808 (serotype VIII) were diluted and incubated with serum at 37°C for 30 min, and the amounts of L-ficolin remaining in the supernatant were estimated. The data are the means ± standard deviations of three experiments. (B) Amount of NeuNAc content remaining on and L-ficolin binding to neuraminidase-treated bacteria. The amount of NeuNAc remaining on neuraminidase-treated bacteria was estimated by using bacterial suspensions having an OD600 of 0.60. Using the L-ficolin binding curve for intact bacteria as a standard for each serotype, the relative L-ficolin binding to neuraminidase-treated bacteria was estimated by using bacterial suspensions having an OD600 of 0.60 for serotypes Ib and V, bacterial suspensions having an OD600 of 0.15 for serotypes III and VI, and bacterial suspensions having an OD600 of 0.0375 for serotype VIII. The data are the means ± standard deviations of three experiments in which the NeuNAc concentration and the L-ficolin binding of a suspension of intact bacteria having an OD600 of 0.60 were each defined as 100%. There was no statistical difference between the relative NeuNAc concentration and the relative L-ficolin binding for each serotype (Student's t test).


Figure 7
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  		FIG. 7. (A to C) CPS content expressed as the NeuNAc content of bacterial cells (A), GBPS content of bacterial cells (B), and LTA content of the bacterial cell surface (C) of wild-type serotype III strains 874391 and COH1, unencapsulated isogenic mutants 874391/cps– and COH1-13, and a mutant in which the CPS lacks NeuNAc (COH1-11). Concentrations are expressed as the NeuNAc concentration for CPS, as the GlcNAc concentration for GBPS, and as the phosphorus concentration for LTA. The data are the means ± standard deviations of three experiments. (D) L-ficolin binding to wild-type serotype III strains, unencapsulated isogenic mutants, and a mutant in which the CPS lacks NeuNAc. Bacteria were diluted and incubated with serum at 37°C for 30 min, and the amount of L-ficolin remaining in the supernatant was estimated. The data are the means ± standard deviations of three experiments.


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DISCUSSION
 
GBS CPS are comprised of combinations of five monosaccharides (17-21, 46, 49-51). NeuNAc, glucose, and galactose are found in the CPS of all nine serotypes. GlcNAc is also included in all CPS except serotype VI and VIII CPS. Rhamnose is present only in serotype VIII CPS. GBPS is composed of GlcNAc, rhamnose, galactose, and glucitol (33). The polyglycerophosphate backbone of GBS LTA is substituted with only D-alanine, whereas in many other species D-alanine substituents alternate with GlcNAc (15).

In the present study, MBL did not bind to any GBS examined, whereas L-ficolin bound to GBS cells belonging to many serotypes. The carbohydrate recognition domain of MBL is a classical C-type lectin domain, which exhibits Ca2+-dependent binding, and it binds to all monosaccharides having equatorial 3- and 4-hydroxyl groups, such as mannose, GlcNAc, and glucose (47). In most cases glucose and GlcNAc form the CPS backbone and side chains with β1-3 or β1-4 linkages; the only exception is serotype V CPS, in which glucose is a nonreducing terminal side residue (17-21, 46, 49-51). Presumably, the inability to bind to MBL reflects the absence of free 3- or 4-hydroxyl group in GBS CPS.

L-ficolin binding to GBS cells correlated with the CPS content in serotypes Ib, III (RDP types III-2 and III-3), and V but not with the GBPS content or with the LTA content. Purified soluble CPS and GBPS inhibited L-ficolin binding to serotype Ib, III, V, VI, and VIII bacteria coated onto microtiter wells in a concentration-dependent manner. On the other hand, purified LTA from these serotypes did not inhibit L-ficolin binding to bacteria coated onto microtiter wells, and no L-ficolin binding to purified LTA that had been coated onto microtiter wells was observed, indicating that L-ficolin does not bind to GBS LTA. We suspect that the inability of purified GBS LTA to bind to L-ficolin results from the absence of GlcNAc in GBS LTA (less than 1 nmol GlcNAc per 100 nmol phosphorus in an LTA preparation), a finding consistent with the previous observation by Henneke et al. that LTA from GBS lacks GlcNAc substituents of the glycerophosphate backbone (15).

Inhibition of L-ficolin binding to bacteria by the monosaccharides that comprise CPS and GBPS was measured in order to determine the ligand for L-ficolin. Glucose, galactose, rhamnose, and glucitol did not inhibit L-ficolin binding, indicating that these monosaccharides do not bind to L-ficolin and/or do not act as ligands for L-ficolin on serotype Ib, III, V, VI, and VIII bacterial cell surfaces. L-ficolin binds to the N-acetylated monosaccharide GlcNAc, N-acetylmannosamine, N-acetylgalactosamine, and other acetylated compounds, such as N-acetylcysteine, N-acetylglycine, and acetylcholine (23). Consistent with these binding specificities, NeuNAc and GlcNAc inhibited L-ficolin binding to bacteria belonging to every serotype, with the exception that GlcNAc did not inhibit L-ficolin binding to serotype VIII bacteria. L-ficolin has multiple sugar binding sites on its globular heads (12). Given that there is no sugar other than NeuNAc and GlcNAc on the GBS cell surface that acts as a ligand for L-ficolin and that NeuNAc and GlcNAc bind preferentially to different sites on the globular heads, these observations suggest that NeuNAc, but not GlcNAc, is a ligand for L-ficolin on the surface of serotype VIII bacteria. NeuNAc inhibited L-ficolin binding to serotype Ib, III, V, and VI bacteria coated onto microtiter wells, and the IC50s of NeuNAc were five- to eightfold lower than those of GlcNAc. These observations suggest that in these serotypes more NeuNAc than GlcNAc is available as ligands for L-ficolin on the cell surface. An alternative explanation is that NeuNAc has a higher affinity than GlcNAc for both of the ligand-binding sites on the globular heads. When NeuNAc was selectively removed from GBS cells by treatment with neuraminidase, the reduction in L-ficolin binding correlated with the amount of NeuNAc removed, suggesting that NeuNAc is the major ligand for L-ficolin on the intact cell surface. This conclusion was strengthened by additional experiments using isogenic mutants of serotype III strains. L-ficolin was able to bind to wild-type strains, but it bound only weakly to unencapsulated mutants and a mutant strain in which the CPS lacks NeuNAc, despite the fact that the mutants expressed larger amounts of LTA than their parent strains and amounts of GBPS similar to the amounts expressed by the parent strains. These data support our assertion that L-ficolin binds to intact GBS surfaces primarily through an interaction with the NeuNAc of CPS. This conclusion is consistent with the previously published observation that L-ficolin does not bind to serotype 14 Streptococcus pneumoniae (24), which expresses a CPS that is structurally identical to a NeuNAc-deficient serotype III CPS (22).

L-ficolin has been shown to bind to fungal cell wall 1,3-β-D-glucan (30). Recently, the recognition region of L-ficolin was solved by X-ray crystallography, and the recognition region interacts not only with acetylated compounds but also with a linear oligosaccharide, β-D-glucan, composed of four glucose units linked through covalent β1-3 bonds. The characteristics of L-ficolin binding to β-D-glucan suggest that L-ficolin binding to CPS also occurs through an interaction with elongated structures in CPS composed of β1-3 or β1-4 linkages (12). CPS appears to bind with a higher affinity to L-ficolin than to NeuNAc and GlcNAc, as estimated by IC50, suggesting that L-ficolin binding is facilitated in polysaccharides in which the ligand is present as repeating units or that L-ficolin binds synergistically to NeuNAc, GlcNAc, and elongated structures with β1-3 or β1-4 linkages.

The hypothesis that L-ficolin binds CPS largely through an interaction with the NeuNAc of CPS, however, is inconsistent with our observation that L-ficolin did not bind to strains of every serotype despite the fact that all GBS CPS contain NeuNAc. L-ficolin bound significantly to all serotype Ib, V, VI, and VIII strains and to RDP type III-2 and III-3 strains, but it did not bind to serotype Ia and II strains or to RDP type III-1 and III-4 strains. The reason why L-ficolin cannot bind to some serotypes or RDP types of GBS that possess a NeuNAc-containing CPS is not clear. GBS CPS are known to modify NeuNAc residues by O acetylation of 5 to 55% of the total NeuNAc, and NaOH treatment eliminates this modification (26). In this study we did not determine the level of O acetylation in the purified CPS preparations. The IC50s for all type-specific CPS that had not been treated with NaOH were almost identical to the IC50s for type-specific CPS that had been treated with NaOH, but the results of these experiments are difficult to interpret because the CPS that had not been treated with NaOH was contaminated with GBPS. However, it is still possible that the variable binding to intact cells of different serotypes could be explained by differences in L-ficolin binding to native and O-acetylated residues since the level of O acetylation varies in different strains (26). It is also possible that the accessibility of ligands to L-ficolin on the bacterial surface differs depending on the serotype and RDP type.

All strains to which L-ficolin/MASP complexes bound were able to consume C4 following incubation in serum under conditions that would prevent formation of the classical pathway C4 convertase by C1. Under these conditions, C4 consumption appeared to be due to cleavage of C4 by L-ficolin/MASP complexes since MBL did not bind to any GBS. MASP circulate in complexes with L-ficolin as an inactive single-chain proenzyme (11). When the complexes bind to a ligand, MASP-2 is cleaved to become an active enzyme, and it activates C4 and C2 to generate C4b2a. Although L-ficolin/MASP complex-dependent C4 consumption was observed for every strain, there was no correlation between the amount of C4 consumption and the amount of L-ficolin binding by different bacteria. The mechanism by which the MASP-2 proenzyme is cleaved to become an active enzyme has not been fully elucidated, but L-ficolin binding to a ligand is required (11). One possible explanation is that cell surface structure affects the three-dimensional structure of L-ficolin bound to cells and affects the cleavage of the proenzyme to the active form.

We previously demonstrated that binding of L-ficolin/MASP complexes to serotype III GBS generates the C3 convertase C4b2a, which cleaves C3 and results in deposition of C3b on GBS (3). C3b deposited by this lectin pathway forms the alternative pathway C3 convertase C3bBb. C3bBb activity is enhanced by CPS-specific IgG2, leading to increased opsonophagocytic killing by further deposition of C3b on the GBS. Bacterium-bound L-ficolin/MASP complexes in such studies may also result in more efficient ingestion and killing of the bacteria by polymorphonuclear leukocytes both in the presence and in the absence of CPS-specific IgG2. Another important function of L-ficolin/MASP complexes in GBS infection may be their action on the ensuing adaptive immune response. GBS release CPS into extracellular fluid, and, as shown here, L-ficolin/MASP complexes can bind to free CPS, even when binding to cell surface CPS is limited. It is possible that complement bound to CPS by the action of L-ficolin/MASP complexes also plays a role in antigen recognition by cells involved in the adaptive immune response.

Recently, Hummelshoj et al. have described polymorphisms within the promoter region and exons of L-ficolin that are associated with marked alterations in L-ficolin serum concentrations and with ligand binding, respectively (16). Further analysis of these polymorphisms and age-associated variations in L-ficolin serum concentrations in GBS-infected and uninfected infants should help us to determine the role of L-ficolin in preventing neonatal GBS infection.


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ACKNOWLEDGMENTS
 
We are grateful to K. Doran of the University of California for helpful discussions.

This study was supported by The Promotion Corporation for Private School of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Microbiology, Joshi-Eiyoh University, Sakado, Saitama 350-0288, Japan. Phone and fax: 81 49 282 3716. E-mail: tshinji{at}eiyo.ac.jp Back

{triangledown} Published ahead of print on 15 October 2007. Back

Editor: F. C. Fang


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Infection and Immunity, January 2008, p. 179-188, Vol. 76, No. 1
0019-9567/08/$08.00+0     doi:10.1128/IAI.00837-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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