M-ficolin binds selectively to the capsular polysaccharides of Streptococcus pneumoniae serotypes 19B and 19C and of a Streptococcus mitis strain

Abstract

The three human ficolins (H-, L-, and M-ficolins) and mannan-binding lectin are pattern recognition molecules of the innate immune system mediating activation of the lectin pathway of the complement system. These four human proteins bind to some microorganisms and may be involved in the resolution of infections. We investigated binding selectivity by examining the binding of M-ficolin to a panel of more than 100 different streptococcal strains (Streptococcus pneumoniae and Streptococcus mitis), each expressing distinct polysaccharide structures. M-ficolin binding was observed for three strains only: strains of the pneumococcal serotypes 19B and 19C and a single S. mitis strain expressing a similar polysaccharide structure. The bound M-ficolin, in association with MASP-2, mediated the cleavage of complement factor C4. Binding to the bacteria was inhibitable by N-acetylglucosamine, indicating that the interaction with the bacterial surface takes place via the fibrinogen-like domain. The common N-acetylmannosamine residue present in the structures of the four capsular polysaccharides of group 19 is linked via a phosphodiester bond. This residue is apparently not a ligand for M-ficolin, since the lectin binds to two of the group 19 polysaccharides only. M-ficolin bound strongly to serotype 19B and 19C polysaccharides. In contrast to those of serotypes 19A and 19F, serotype 19B and 19C polysaccharides contain an extra N-acetylmannosamine residue linked via glycoside linkage only. Thus, this extra residue seems to be the M-ficolin ligand. In conclusion, we were able to demonstrate specific binding of M-ficolin to some capsular polysaccharides of the opportunistic pathogen S. pneumoniae and of the commensal bacterium S. mitis.

Fig 1

Binding of M-ficolin to S. pneumoniae. Formalin-fixed bacteria were first incubated with 10% NHS and then spun down, and the M-ficolin remaining in the supernatant was measured. The percentage of bound M-ficolin was calculated based on the concentration in the supernatants compared to the concentration in 10% NHS. The designations of the serotypes are given on the x axis. Data are representative of two individual experiments. Means and standard deviations are shown.

Fig 2

Binding of PRMs to S. pneumoniae. (A to D) Formalin-fixed pneumococci (10⁹) of selected serotypes were incubated with 400 μl 10% NHS, and the lectin pathway PRMs remaining in the supernatant after the removal of the bacteria by centrifugation were measured. The percentage of bound PRMs was calculated based on the concentration in the supernatants compared to the concentration in 10% NHS. (A) M-ficolin binding; (B) MBL binding; (C) H-ficolin binding; (D) L-ficolin binding. The designations of the serotypes are given on the x axis. Data are representative of two individual experiments. Means and standard deviations are shown. (E) Concentration-dependent binding of M-ficolin to cells of pneumococcal serotypes 19B and 19C. Data are representative of two individual experiments.

Fig 3

Inhibition of binding of M-ficolin to S. pneumoniae strains of serotypes 19B and 19C. (A) The binding of serum M-ficolin to 19B and 19C strains (in the presence of 100 mM GlcNac or 100 mM glucose) and to proteinase K-treated bacteria was tested as described for Fig. 2. The data are presented as percentages of the amount of serum M-ficolin that was bound if no carbohydrate was added and are representative of two individual experiments. Means and standard deviations are shown. (B and C) The binding of rM-ficolin to microtiter wells coated with pneumococcal cells of serotype 19B (B) or 19C (C) was tested in the presence of different monosaccharides or purified 19C or 19F capsular polysaccharide (CPS). Data are given as counts per second on the y axis and are representative of two individual experiments. The amounts of the various potential inhibitors used are given in millimolar concentrations for the monosaccharides and in micrograms per milliliter for the capsular polysaccharides.

Fig 4

Complement deposition on S. pneumoniae by rM-ficolin/rMASP-2 complexes. (A) rM-ficolin was incubated in wells coated with cells of serotype 19B, 19C, and 19F strains. Bound rM-ficolin was detected with an anti-M-ficolin antibody. (B) In parallel wells in which rMASP-2 was associated with the bound rM-ficolin, complement activation was probed by adding C4, incubating at 37°C, and then detecting the C4b deposited. Data are representative of two individual experiments.

Fig 5

Flow cytometric and Western blot analyses of the binding of M-ficolin to S. pneumoniae. (A) Binding of rM-ficolin to serotype 19B (dotted line), 19C (solid line), and 19F (dashed line) strains. (B) Inhibition of the binding of rM-ficolin to a serotype 19C strain by GlcNAc (dashed line) or glucose (solid line). Shaded curves represent rM-ficolin-treated 19C cells with no anti-M-ficolin antibody added (negative control). Isotype controls [IgG1(κ)] gave results identical to those represented by the shaded curves. Data are representative of two individual experiments. (C) Western blot analysis. M-ficolin bound to pneumococcal 19F, 19B, or 19C cells after incubation with NHS and washing was eluted with GlcNAc, and the eluates were examined by SDS-PAGE under nonreducing conditions, followed by blotting onto a nitrocellulose membrane and detection with a monoclonal anti-M-ficolin antibody. Data are representative of two individual experiments. Molecular markers are shown on the right.

Fig 6

Binding of ficolins and MBL to S. mitis. The binding of the lectin pathway PRMs was assessed as described in the legend to Fig. 1. (A) M-ficolin; (B) MBL; (C) H-ficolin; (D) L-ficolin. The designations of the strains are given on the x axis. Data are representative of two individual experiments. Means and standard deviations are shown.

Fig 7

Capsular structures of S. pneumoniae serogroup 19. The repeating unit of the four capsular polysaccharides of serogroup 19 is shown. Serotype 19B and 19C polysaccharides differ from serotype 19A and 19F polysaccharides by having an extra ManNAc residue with a branched terminal d-ribose linked to l-rhamnose. In addition, the capsular polysaccharide of serotype 19C also has a d-glucose residue linked to the extra ManNAc residue.