Deciphering the molecular bases of Mycobacterium tuberculosis binding to the lectin DC-SIGN reveals an underestimated complexity

Abstract

Interactions between dendritic cells and Mycobacterium tuberculosis, the aetiological agent of tuberculosis in humans, are thought to be central to anti-mycobacterial immunity. We have previously shown that M. tuberculosis binds to human monocyte-derived dendritic cells mostly through the C-type lectin DC-SIGN (dendritic-cell-specific intercellular molecule-3-grabbing non-integrin)/CD209, and we have suggested that DC-SIGN may discriminate between mycobacterial species through recognition of the mannose-capping residues on the lipoglycan lipoarabinomannan of the bacterial envelope. Here, using a variety of fast- and slow-growing Mycobacterium species, we provide further evidence that mycobacteria recognition by DC-SIGN may be restricted to species of the M. tuberculosis complex. Fine analyses of the lipoarabinomannan molecules purified from these species show that the structure and amount of these molecules alone cannot account for such a preferential recognition. We propose that M. tuberculosis recognition by DC-SIGN relies on both a potential difference of accessibility of lipoarabinomannan in its envelope and, more probably, on the binding of additional ligands, possibly including lipomannan, mannose-capped arabinomannan, as well as the mannosylated 19 kDa and 45 kDa [Apa (alanine/proline-rich antigen)] glycoproteins. Altogether, our results reveal that the molecular basis of M. tuberculosis binding to DC-SIGN is more complicated than previously thought and provides further insight into the mechanisms of M. tuberculosis recognition by the immune system.

Figure 1. DC-SIGN has high affinity for species of the M. tuberculosis complex

(A) Phylogenetic tree of the mycobacteria species used in the present study, based on partial 16 S RNA sequences. (B) Epithelial HeLa-derived cells expressing or not DC-SIGN (HeLa::DC-SIGN and HeLa respectively) were infected with fast-growing (M. chelonae, M. smegmatis, M. fortuitum), slow-growing non-TB (M. marinum, M. kansasii, M. avium, M. xenopi, M. gordonae) or TB complex (M. tuberculosis H37Rv, M. bovis, M. bovis BCG, and M. africanum) species at a multiplicity of infection of 1 bacterium/cell. Bacterial binding was evaluated after 4 h at 4 °C by counting colony-forming units. Results are means (±S.D.) of the ratio between binding to HeLa::DC-SIGN and binding to HeLa for three to five independent experiments. A ratio of 1 (dotted line) indicates no binding to the lectin.

Figure 2. ManLAMs of M. tuberculosis H37Rv, M. bovis BCG, M. avium, M. xenopi, M. marinum and M. kansasii have similar mannose caps contents

Mannose caps were analysed and quantified by CE–LIF after mild acid hydrolysis of LAM and APTS derivatization as previously described [23]. M, α-D-Manp; MM, α-D-Manp-(1→2)-α-D-Manp; MMM, α-D-Manp-(1→2)-α-D-Manp-(1→2)-α-D-Manp.

Figure 3. ManLAMs of non-TB slow-growing mycobacteria can inhibit M. tuberculosis binding to DC-SIGN

HeLa or HeLa::DC-SIGN cells were preincubated with 10 μg/ml ManLAM from M. tuberculosis, M. xenopi, M. kansasii, M. avium or M. marinum, or saline (Ø) for 1 h at 4 °C and infected with M. tuberculosis H37Rv as described in Figure 1. Bacteria binding was measured as described in Figure 1. Data are expressed as percentages of binding relative to control values (100%, preincubation of HeLa::DC-SIGN cells with saline), and the means (±S.D.) for three independent experiments are shown. P values are given as assessed by Student's t test comparison with HeLa cells. NS, not significant.

Figure 4. Analyses of lipoglycans from control and biotin hydrazide-labelled M. bovis BCG and M. xenopi

(A) Periodic acid/AgNO₃ staining of SDS/PAGE gel. (B) Western blot probed with alkaline phosphatase-conjugated avidin. Lanes 1 and 9, biotinylated BSA standard (0.1 μg); lanes 2 and 8, in vitro biotinylated M. bovis BCG LAM and LM standards (5 μg); lanes 3 and 4, lipoglycans from biotin hydrazide-labelled and control M. bovis BCG respectively (8 μg); lane 5, in vitro biotinylated M. xenopi LAM standard (5 μg); lanes 6 and 7, lipoglycans from biotin hydrazide-labelled and control M. xenopi respectively (8 μg). (C) Quantification of biotin hydrazide labelling by ELISA. Two independent experiments of labelling were performed on both M. bovis BCG and M. xenopi. Portions (300 ng) of lipoglycans from labelled and control bacteria were adsorbed on the microtitre wells and probed with alkaline phosphatase-conjugated avidin. The absorbance values are given without correction (I) or corrected by the response to the monoclonal CS-35 anti-LAM antibody (II) or by the response to a rabbit anti-BCG serum (III). Abbreviation: a.u., absorbance units.

Figure 5. Flow-cytometric analysis of M. bovis BCG and M. xenopi labelling with the monoclonal CS-35 anti-LAM antibody

M. bovis BCG (A) or M. xenopi (B) were incubated with anti-LAM monoclonal antibody CS-35, which was revealed by fluorophore-Cy3-conjugated anti-mouse IgG antibody and analysed by flow cytometry (‘Labelled’). Analysis was carried on 10000 bacteria. Controls consisted of unlabelled bacteria (‘Unlabelled’) and bacteria incubated with secondary antibody only (‘2^nd Ab’). ΔMFI, mean fluorescence intensity variation between CS-35-labelled bacteria and bacteria incubated with secondary antibody only. The Figure is representative of two independent experiments.

Figure 6. Other non-ManLAM M. tuberculosis-derived ligands can inhibit M. tuberculosis binding to DC-SIGN

(A) HeLa or HeLa::DC-SIGN cells were preincubated with 10 μg/ml M. tuberculosis-derived ManLAM, LM, ManAM, 19 kDa antigen, 45 kDa antigen or saline (Ø) for 1 h at 4 °C and infected with M. tuberculosis H37Rv as described in Figure 1. Bacteria binding was measured as described in Figure 1. Data are expressed as described in Figure 3 and represent the means (±S.D.) for three independent experiments. P values are given as assessed by a Student's t test comparison with HeLa cells. (B) HeLa::DC-SIGN and HeLa cells were infected with 45 kDa-protein-null (two strains) or 19 kDa-protein-null (one strain) mutants of M. tuberculosis, or with their wild-type counterparts (H37Rv1 and H37Rv2). Bacterial binding was evaluated as described in Figure 1. Data are expressed as described in Figure 1 and represent the means (±S.D.) for three independent experiments. P values are given as assessed by a Student's t test comparison with wild-type strains. NS, not significant. (C) HeLa::DC-SIGN and HeLa cells were infected with 45 kDa- or 19 kDa-protein-expressing recombinant M. smegmatis (MS) or the wild-type strain. Bacterial binding was evaluated as described in Figure 1. Data are expressed as described in Figure 1 and represent the means (±S.D.) for three independent experiments. P values are given as assessed by a Student's t test comparison with the wild-type strain. NS, not significant.