Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition

Abstract

Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. Diverse Candida species, Aspergillus fumigatus, Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereas Escherichia coli, Klebsiella species, and Haemophilus influenzae type b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci, Streptococcus pneumoniae, and Staphylococcus epidermidis showed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.

FIG. 1

Representative population density plots of three organisms (S. aureus NCTC6571, C. lusitanae, and E. faecalis) detected by a combination of forward scatter and side scatter and gated as regions R1, R2, and R3, respectively (a to c). The corresponding flow cytometric profiles of MBL binding to these organisms using the protein at a final concentration of 5 μg/ml are shown (d to f). The control profiles (shaded) show organisms incubated with FITC-conjugated anti-MBL alone. Experimental samples (open profiles) were obtained after incubation of organisms with MBL and FITC-conjugated anti-MBL.

FIG. 2

Representative cytometric profile of MBL binding to K. aerogenes (a). A typical tail is seen in the experimental sample, shown as an open profile, but not in the control profile (shaded). The forward scatter-versus-side scatter density plot of the entire population (b) is shown. The size and granularity characteristics of the subpopulation (c) did not differ markedly from those shown in panel b.

FIG. 3

MBL binding to S. aureus isolates ranked in order of binding. Results are expressed as median fluorescence, with each bar representing the mean of three to six experiments; error bars, standard errors of the means. Solid bars, clinical isolates; open bars, defined strains.

FIG. 4

MBL binding to S. aureus NCTC6571 at different concentrations. Binding of MBL is expressed as median fluorescence. Each point represents the mean of three experiments ± the standard error of the mean. Both structural gene mutations and promoter polymorphisms determine the serum MBL level and may influence the degree of MBL binding to the organisms in vivo. The approximate MBL concentration ranges of individuals with and without mutations are indicated by arrows.

FIG. 5

Inhibition of MBL binding to six representative microorganisms using two monosaccharides (solid line, d-mannose; dotted line, N-acetylglucosamine) at different concentrations and 10 mM EDTA (■). The monosaccharide or EDTA was added to the MBL solution 10 min prior to the addition of the MBL to the organisms. Each point represents the mean ± standard error of the mean of the MFI for three independent experiments.

FIG. 6

MBL binding to 80 microorganisms. Data are expressed as the ratios of median fluorescence of the experimental samples (incubated with MBL and FITC-conjugated anti-MBL) to the median fluorescence of the control samples (incubated with FITC-conjugated anti-MBL alone). Each circle represents the mean of three to six experiments. The solid circles represent clinical isolates, whereas the open circles show either defined National Collection of Type Cultures strains or internal quality controls. The dashed line represents a ratio of one. β-haem. Strep. A indicates beta-hemolytic group A streptococcus.

FIG. 7

C4 deposition on three representative organisms previously incubated with different concentrations of MBL. S. aureus NCTC6571 (●) and one strain of Klebsiella (▴) were selected for their known ability to bind MBL, whereas another strain of Klebsiella (⧫) did not bind to the protein. Each point represents the mean ± the standard error of the mean of the MFI for three independent experiments.