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Bactericidal/Permeability-Increasing Protein Is an Enhancer of Bacterial Lipoprotein Recognition

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Bactericidal/Permeability-Increasing Protein Is an Enhancer of Bacterial Lipoprotein Recognition

Sigrid Bülow et al. Front Immunol.

Abstract

Adequate perception of immunologically important pathogen-associated molecular patterns like lipopolysaccharide and bacterial lipoproteins is essential for efficient innate and adaptive immune responses. In the context of Gram-negative infection, bactericidal/permeability-increasing protein (BPI) neutralizes endotoxic activity of lipopolysaccharides, and thus prohibits hyperactivation. So far, no immunological function of BPI has been described in Gram-positive infections. Here, we show a significant elevation of BPI in Gram-positive meningitis and, surprisingly, a positive correlation between BPI and pro-inflammatory markers like TNFα. To clarify the underlying mechanisms, we identify BPI ligands of Gram-positive origin, specifically bacterial lipopeptides and lipoteichoic acids, and determine essential structural motifs for this interaction. Importantly, the interaction of BPI with these newly defined ligands significantly enhances the immune response in peripheral blood mononuclear cells (PBMCs) mediated by Gram-positive bacteria, and thereby ensures their sensitive perception. In conclusion, we define BPI as an immune enhancing pattern recognition molecule in Gram-positive infections.

Keywords: Gram-positive; Streptococcus pneumoniae; bacterial lipoprotein; bactericidal/permeability-increasing protein; lipoteichoic acid; pattern recognition; pro-inflammatory.

Figures

Figure 1
Figure 1
Correlation of BPI with pro-inflammatory markers in Gram-positive meningitis. BPI was measured in CSF of patients with bacterial meningitis caused by Streptococcus pneumoniae (SP, n = 13), Neisseria meningitidis (NM, n = 7) and enterovirus-infected controls (EnV, n = 20). (A) Results are expressed as means ± SEM. Statistics for comparison of the difference of the BPI level were performed with the unpaired Student's t-test (p-values are indicated). Correlation of BPI with TNFα and IL-6 (B) and LBP (C) as well as correlation of LBP with TNFα and IL-6 (D) are depicted. Correlation was analyzed using Pearson's correlation (r- and p-values are indicated). Logarithmic values were used for statistical testing.
Figure 2
Figure 2
Competition of bLPs with lipopolysaccharide for binding to BPI. BPI binding assays with LPS biotin (A–C,F–H) or Pam2CSK4 biotin-coated plates (D,E). “unc.” shows binding of rBPI in uncoated wells treated otherwise identically (A,B). rBPI was pre-incubated with increasing concentrations of LPS EC (A,D), the racemate Pam3CSK4 (B), (R)-Pam3CSK4 (C,E) and (S)-Pam3CSK4 (C) or peptidoglycan of S. aureus (PGN SA; F). Furthermore, pre-incubations of rBPI with different heat-inactivated bacterial lysates are shown (G). Preparations of rBPI and neutrophil BPI of two different sources [BPIN(W) and BPIN(A)] were pre-incubated with (R)-Pam3CSK4 (H). Absorbance measured at 450 nm for wells with BPI alone was set to 100% to ensure comparability between the different ligands. All results are shown as means ± SD of three biological replicates.
Figure 3
Figure 3
Determination of the affinity of BPI to bLPs. MST binding assay (A–D). Changes in movement in MST were monitored using BPIN(A) NT647 incubated with increasing concentrations of the ligands [LPS EC (A), Pam3CSK4 (C)] or Pam3CSK4 Fluorescein incubated with increasing concentrations of BPIN(A) (D). The binding affinity and r2 values are indicated. The same assay using NT647-labeled rBPI was used to calculate KD values for the interactions of the protein to LPS EC, LPS EC BL21, and (R)-Pam3CSK4 (B). NanoDSF was performed for rBPI incubated with the indicated ligands (E,F). Temperature-dependent change in fluorescence is indicated for the wavelength of 350 nm [E, upper part: absolute values, lower part: first derivate (f')]. Vertical lines indicate Tm. The shift in melting temperature (Tshift) caused by the ligands is shown (F). Temperature shifts above 1°C are interpreted as the influence of an interaction on the thermal stability of the protein. Data represent the mean of two (A,C,D) or three (B) technical replicates or means ± SD of two biological replicates (E,F).
Figure 4
Figure 4
Structural requirements for binding of bLPs to BPI. BPI binding assay (A–F). Lipopeptides with modified structure as well as DAG were tested for their potential to inhibit binding of rBPI to LPS biotin-coated wells (A–F). Absorbance measured at 450 nm for wells with rBPI alone was set to 100% to ensure comparability between the different ligands. All results are shown as means ± SD of at least four biological replicates. R1 = CH3(CH2)14. Electrostatic charge surface of the BPI structure (G). N-terminal lipid-binding pocket surrounded by differently charged areas is depicted with phosphatidylcholine as ligand.
Figure 5
Figure 5
LTA is an additional ligand of BPI in Gram-positive bacteria. BPI binding assay (AE, G,H). MST binding assay (F,I). Lysates of S. pneumoniae D39Δcps, D39ΔcpsΔlgt (SPΔlgt), D39ΔcpsΔlsp (SPΔlsp) and D39ΔcpsΔlgtΔlsp (SPΔlgtΔlsp; 50 μg/ml) were incubated with rBPI to evaluate their potential to inhibit the binding to LPS biotin-coated wells (A, NT: not treated). Statistics for comparison were performed with the unpaired Student's t-test (p-value *p = 0.004). LTA SP and LTA SA were tested for their potential to inhibit the binding of rBPI to LPS biotin-coated wells (B,G). To correct for the effects of contaminating bLPs, LTAs of the corresponding Δlgt strains were tested (C,H). Data for LTA SPΔlgt-N2H4 as well as WTA SPΔlgt are shown (D,E). Absorbance measured at 450 nm for wells with rBPI alone was set to 100% to ensure comparability between the different ligands. Results are shown as means ± SD of three (BE, G,H) or four (A) biological replicates. rBPI NT647 was monitored in MST when incubated with increasing concentrations of LTA SPΔlgt and WTA SPΔlgt (F) as well as increasing concentrations of LTA SAΔlgt (I). KD and r2 values are indicated for LTA SPΔlgt and LTA SAΔlgt (F,I). Data represent the mean of three technical replicates (F,I).
Figure 6
Figure 6
Influence of BPI on TNFα secretion of PBMCs in response to bLPs and lysates of S. pneumoniae D39Δcps. TNFα-ELISA of the supernatants of PBMCs stimulated with TLR-ligands [LPS EC, (R)-Pam3CSK4 or (R)-FSL-1] ± rBPI at the indicated concentrations for 18 h (A). Independent titration experiments performed in four different donors are indicated as relative increase compared to cytokine secretion with TLR ligand alone (A). The relative increase in TNFα, IL-6, and IL-8 in the supernatants of PBMCs stimulated with (R)-Pam3CSK4 ± rBPI is represented (C). One donor (•) is not shown in (C) for stimulation since IL-6 was beyond the linear range of the ELISA. The relative increase in TNFα in the supernatants of PBMCs stimulated with (R)-Pam3CSK4 ± BPIN(A) or LTA SP ± rBPI is summarized (B,D). PBMCs were stimulated with lysates of S. pneumoniae D39Δcps and S. aureus 113 and the relative change in secreted TNFα, IL-6, and IL-8 caused by the addition of BPI is shown (E,F). Results of independent stimulations of PBMCs of four (A,D) or seven (B,C,E,F) different donors are shown. Each symbol represents an individual donor (B–F). Unless otherwise indicated, the BPI concentration used was 500 nM. Statistics for comparison of the relative cytokine secretion ± BPI were performed with the paired Student's t-test (p-values are indicated, BF). Results are shown as means ± SEM (A).

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