Bacterial lectin BambL acts as a B cell superantigen

Abstract

B cell superantigens crosslink conserved domains of B cell receptors (BCRs) and cause dysregulated, polyclonal B cell activation irrespective of normal BCR-antigen complementarity. The cells typically succumb to activation-induced cell death, which can impede the adaptive immune response and favor infection. In the present study, we demonstrate that the fucose-binding lectin of Burkholderia ambifaria, BambL, bears functional resemblance to B cell superantigens. By engaging surface glycans, the bacterial lectin activated human peripheral blood B cells, which manifested in the surface expression of CD69, CD54 and CD86 but became increasingly cytotoxic at higher concentrations. The effects were sensitive to BCR pathway inhibitors and excess fucose, which corroborates a glycan-driven mode of action. Interactome analyses in a model cell line suggest BambL binds directly to glycans of the BCR and regulatory coreceptors. In vitro, BambL triggered BCR signaling and induced CD19 internalization and degradation. Owing to the lectin's six binding sites, we propose a BCR activation model in which BambL functions as a clustering hub for receptor glycans, modulates normal BCR regulation, and induces cell death through exhaustive activation.

Keywords: Adaptive immunity; Apoptosis; Bacterial pathogens; Immunoglobulin glycosylation; Multivalent lectins; β-Propeller lectins.

Fig. 1

Structure comparison of B cell-activating and BCR-binding lectins. BambL (from Burkholderia ambifaria, PDB entry 3ZWE), LecB (Pseudomonas aeruginosa, 5A6X), Bc2L-A (B. cenocepacia, 4AOC), and hemagglutinin (H5N1 influenza virus, 2FK0). Proteins are rendered with PyMOL as surface presentations, sugars as sticks, and calcium ions in the binding pockets of LecB and Bc2L-A as green spheres. One monomer per lectin is colored blue. Arrows and numbers mark the approximate distances between individual sugar binding sites. BambL, although the smallest in size, has by far the highest valency

Fig. 2

BambL activates peripheral human B cells but becomes cytotoxic at increasing concentrations. B cells were enriched from peripheral blood mononuclear cells (PBMCs) and cultivated in the presence of three different BambL concentrations or left unstimulated for 4 days (n = 1 for each data point). Samples were analyzed by flow cytometry after each day. a BambL is cytotoxic to B cells. Histograms of DAPI fluorescence (first row) and light scattering plots (second row) of ungated cells after 16 h of lectin exposure demonstrate that 0.1 µg/mL BambL is tolerated well, but higher concentrations become increasingly cytotoxic. b BambL stimulates surface CD69 expression. Viable B cells (DAPI⁻ CD19⁺) were identified in the lymphocytes gate in a and analyzed for surface CD69 fluorescence. Histograms depict the time course over 4 days. c All B cell subsets are affected by BambL. Viable naive (‘N’, IgD⁺ CD27⁻), marginal‑zone (‘MZ’, IgD⁺ CD27⁺) and class-switched memory (‘SM’, IgD⁻ CD27⁺) B cells were identified in samples after 16 h lectin exposure (scatter plots in first row, numbers in quadrants represent subset proportions). Surface CD69 fluorescence intensities are presented as histograms in the second row. While all subsets experienced an increase in surface CD69, the naive cells displayed the strongest response

Fig. 3

BambL stimulates the expression of classical activation markers, which is sensitive to inhibition of SYK, PI3K and ERK1/2. PBMCs were treated overnight and analyzed by flow cytometry. Viable B cells (DAPI⁻ CD19⁺) and B cell subsets were identified within the samples after analysis as before. Bars are mean fold changes of signal-positive cell frequencies relative to unstimulated controls (error bars are SD). Statistical significance (ANOVA with Tukey’s correction): ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001). a BambL stimulates the expression of B cell activation markers. PBMCs (n = 9–10) were cultivated for 16 h in the presence of BambL, anti-IgM or left unstimulated. BambL elicited an upregulation of CD54, CD69 and CD86, which was most prominent in the naive B cell subset. b Inhibition of intracellular pathways diminishes B cell activation by BambL. B cells (n = 6–9) were first enriched from PBMCs and pre-treated with inhibitors against PI3K, SYK or ERK1/2 for 16 h (‘DMSO’: solvent controls), then stimulated and analyzed for surface expression of CD69 as before. c Excess fucose prevents cell death and activation. PBMCs (n = 9) were stimulated as in a, but 1 µg/mL BambL was included as an additional, cytotoxic stimulus in this experiment. A second set of treatment solutions was supplemented with L-fucose prior to the experiment (‘ + fuc’). The first panel shows the relative frequencies of all DAPI-positive cells (ungated PBMCs). Herein, viable B cells were identified and analyzed for their surface CD69 expression as before (second panel and subset panels). Samples exposed to 1 µg/mL lectin were excluded from the CD69 analyses because of their magnitude of cell death

Fig. 4

BambL extracts the BCR and regulatory coreceptors from Ramos cells and shows direct interaction in far-western lectin blots. a SILAC-based, mass-spectrometric screen of the BambL surface interactome on Ramos cells. BambL-biotin was allowed to bind to surface glycans only, and then the lectin-receptor complexes were extracted and identified my MS/MS peptide fingerprinting. The peptide hits enriched by BambL-biotin versus the negative control (BambL) of two experiments are plotted on a log₂ fold-change scale. Peptides enriched ≥ fourfold were considered significant. Prominent hits are highlighted in the plot and listed in the table below (plot and table share the same color code). b Western blot validation of selected screening hits. Surface proteins were extracted with BambL-biotin as before. Whole-cell lysate (= pull-down input, ‘in’) and extract (‘ex’) were resolved by SDS-PAGE with different acrylamide concentrations (amount of loaded input ≙ 2% of extract amount). In control experiments, BambL-biotin was blocked with excess L-fucose. The sample succession in the images of CD45 and CD22 was originally swapped. These images were re-composed to maintain a consistent sample order across the presentation. c Far-western lectin blots identify direct binding partners of BambL. Pull-down input and extract were resolved by SDS-PAGE and transferred onto a membrane as before, then incubated with fluorescently labeled BambL (BambL-700). After washing, discrete bands were visible in the 700-nm channel (left image) which could be counter-stained in classical immunoblots in the next step (developed with fluorescent secondary antibodies in the 800-nm channel). Images look different because the staining and imaging procedure was individually optimized for each target. d Deglycosylated proteins no longer bind BambL. Whole-cell lysate was treated with the glycosidase PNGase F prior to the lectin blot to remove N-linked glycans. Glycosidase-treated proteins migrated farther (the µHC double band merged into a single band) and the lectin signal was almost absent from these sample lanes

Fig. 5

BambL induces BCR signaling. a Simplified scheme of intracellular BCR signaling. The protein phosphorylation of colored pathway members was used as a readout of their activation state (same color code applies to (b). b BambL induces phosphorylation of SYK, CD19, AKT and ERK1/2. Ramos cells were exposed to BambL for 5, 10 or 30 min, lysed and analyzed in western blots. As controls, cells were either left unstimulated (‘ctrl’) or supplemented with excess L-fucose and stimulated for 30 min (‘30 + fuc’). Phosphorylation-specific signal intensities (‘phospho’) were first normalized to their respective total proteins (‘pan’), then to their 10‑min sample (internal reference per experiment). Bars are mean fold changes (n = 3 for pCD19; n = 4 for pSYK, pAKT, pERK). Statistical significance (2-way ANOVA with Dunnett’s correction): ns (p > 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001), **** (p ≤ 0.0001)

Fig. 6

BambL is internalized and depletes surface and total CD19. a B cells internalize BambL. Ramos cells were loaded with BambL-488 (green) on ice, washed and transferred to warm medium to reinitiate internalization processes. Cells were fixed after indicated time points, counter-stained with DAPI (blue) and mounted for confocal microscopy. Representative confocal sections demonstrate a homogenous lectin distribution on the cell surface at time point 0 min, followed by an internalization wave and accumulation near the nucleus within 60 min. Scale bars: 5 µm. b BambL depletes surface CD19. Ramos cells were loaded with BambL on ice, washed and incubated in warm medium for indicated durations until flow cytometric analysis of surface proteins. As controls, cells were left without lectin (‘ctrl’) or BambL was supplemented with excess L-fucose (‘4 h + fuc’). Cells for time point “0 h” were loaded with lectin, washed and then analyzed without incubation in warm medium. Representative fluorescence histograms of four conditions are presented in the upper row. Bars below are mean fold changes of geometric mean fluorescence intensities (MFI) relative to ‘ctrl’ samples (n = 8). Statistical significance (2-way ANOVA with Dunnett’s correction): ns (p > 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001). c B cells internalize CD19 upon BambL stimulation. Transfected BJAB cells expressing CD19-RFP and BAFFR-YFP were stimulated with BambL for 1 h (controls were left unstimulated), then imaged with a widefield microscope. Representative images demonstrate the internalization of both CD19 and BAFFR. d BambL induces degradation of internalized CD19. Ramos cells were incubated with BambL for indicated durations and analyzed in western blots. To control for differences in sample loading, the CD19 band intensities were normalized once to their total protein signal per lane (left image), once to tubulin. Bars are mean fold changes relative to unstimulated controls (n = 5). Either normalization strategy yielded a significant loss of total CD19 abundance over time. Statistical significance (2-way ANOVA with Dunnett’s correction): ns (p > 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001)

Fig. 7

Proposed models of cognate vs. non-cognate and polyclonal B cell activation. a Classical cognate activation by antigens. Arrayed antigens (green) on the surface of a pathogen or an antigen-presenting cell stimulate BCR signaling, expression of activation markers CD69, CD86/80 and CD54, internalization of the BCR-antigen complex, and antigen processing for presentation on MHC-II. A complementary T helper cell (T_h) provides costimulatory signals through CD40L and cytokines such as IL-2, which initiate a B cell differentiation program into antibody-secreting plasma cells. b Proposed model for non-cognate and polyclonal activation. B cell superantigens like SpA crosslink conserved BCR sites, downmodulate BCR and CD19 expression, and pan-activate B cells irrespective of their antigen specificity. We propose multivalent lectins like BambL achieve a similar effect by crosslinking BCR glycans. In the absence of sufficient survival and differentiation signals, the B cells succumb to exhaustion in a form of activation-induced cell death