DataSheet_1_Lipidation of pneumococcal proteins enables activation of human antigen-presenting cells and initiation of an adaptive immune response.pdf

Streptococcus pneumoniae remains a significant global threat, with existing vaccines having important limitations such as restricted serotype coverage and high manufacturing costs. Pneumococcal lipoproteins are emerging as promising vaccine candidates due to their surface exposure and conservation across various serotypes. While prior studies have explored their potential in mice, data in a human context and insights into the impact of the lipid moiety remain limited. In the present study, we examined the immunogenicity of two pneumococcal lipoproteins, DacB and MetQ, both in lipidated and non-lipidated versions, by stimulation of primary human immune cells. Immune responses were assessed by the expression of common surface markers for activation and maturation as well as cytokines released into the supernatant. Our findings indicate that in the case of MetQ lipidation was crucial for activation of human antigen-presenting cells such as dendritic cells and macrophages, while non-lipidated DacB demonstrated an intrinsic potential to induce an innate immune response. Nevertheless, immune responses to both proteins were enhanced by lipidation. Interestingly, following stimulation of dendritic cells with DacB, LipDacB and LipMetQ, cytokine levels of IL-6 and IL-23 were significantly increased, which are implicated in triggering potentially important Th17 cell responses. Furthermore, LipDacB and LipMetQ were able to induce proliferation of CD4+ T cells indicating their potential to induce an adaptive immune response. These findings contribute valuable insights into the immunogenic properties of pneumococcal lipoproteins, emphasizing their potential role in vaccine development against pneumococcal infections.</p

Additional file 4: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Exogenous application of CRAMP reduced NFκB translocation in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT (A) or KO (B) mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 30 min, 1 or 2 h. After incubation cells were fixed and immunolabeled using anti-NFκB p65 antibody (red) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 19383 kb

Additional file 5: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Co-stimulation of exogenous CRAMP and bacterial supernatant NM induced increase of HO-1 immunofluorescence in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT or KO mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 6 h. After incubation cells were fixed and immunolabeled using anti-HO-1 antibody (green) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 3834 kb

The microbial binding site is located within the third HBD.

<p>(A) Schematic representation of the different truncated vitronectin (Vn) fragments and deletion mutants used for analysis of vitronectin binding. (B) Eight different microbes, including Gram-negative and Gram-positive bacteria and <i>C</i>. <i>albicans</i> were incubated with truncated recombinant vitronectin fragments and three vitronectin deletion mutants. NHS was run in parallel as a positive control. Microbes were washed and total proteins were separated by SDS-PAGE gels, transferred to a membrane and bound vitronectin fragments were detected using an anti- vitronectin polyclonal antiserum. One representative experiment of three independent ones performed is shown. (C) Microbes were immobilized on microtiter plates and incubated with vitronectin fragments including deletion mutants. Bound fragments were detected with rabbit anti-vitronectin pAb and secondary HRP-conjugated goat anti-rabbit pAb. The mean values of three experiments are shown with error bars indicating SD. Statistical significance of differences was estimated using Student’s <i>t</i> test. *, <i>p</i>≤ 0.05; **, <i>p</i>≤ 0.01; ***, <i>p</i>≤ 0.001.</p

Pathogenic microbes bind the terminal complement pathway inhibitor vitronectin.

<p>A series of Gram-negative and Gram-positive bacterial species and <i>Candida albicans</i> were grown overnight and incubated with [¹²⁵I]-labeled vitronectin purified from plasma (A) or recombinant vitronectin^80-396 (B). After washing, bound vitronectin was determined by liquid scintillation counting. The mean values of three experiments are shown. In C, the relative binding of [¹²⁵I]-labeled vitronectin to selected microbes are shown. The mean values of three experiments are shown with error bars indicating standard deviations (SD).</p

Additional file 3: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Proliferation and apoptosis induction after bacterial stimulation in CRAMP-WT or CRAMP-KO microglial cells. Microglial cells from CRAMP-knockout (KO) or wild-type (WT) mice were incubated with bacterial supernatants of Gram-positive bacterium Streptococcus pneumoniae (SP) or Gram-negative bacterium Neisseria meningitidis (NM) and bacterial cell wall components lipopolysaccharide (LPS) or peptidoglycan (PGN) for 24 h. After incubation, glial cells were fixed and immunolabeled using the proliferation marker Ki67 (red), TUNEL reaction mixture for apoptosis and DAPI for nuclear counterstaining (blue). (A) Representative results from one of three independent experiments. (B) Ki67 proliferation index was calculated by the number of positive cells expressing Ki67 divided by the total number of cells in each field. These results were calculated for at least 20 separate cells. Scale bar = 20 μm. (TIFF 6059 kb

The binding of vitronectin is inhibited by heparin and high ionic strength.

<p>Heparin and NaCl inhibited the binding of [¹²⁵I]-labeled vitronectin to the microbial pathogens. The microbial pathogens were incubated with [¹²⁵I]-labeled vitronectin and 10 μM heparin (A) or 1 M NaCl (B), followed by washing and determination of radioactivity associated with the pellet. The vitronectin binding of each microbe in the absence of competitor was defined as 100%. The mean values of three experiments are shown with error bars indicating SD. Statistical significance of differences was estimated using Student’s <i>t</i> test. **, <i>p</i>≤ 0.01; ***, <i>p</i>≤ 0.001.</p

Vitronectin bound to intact bacteria inhibits C5b-9 deposition.

<p>Vitronectin (25–50 μg/ml) was bound to Hib (<i>A</i>), NTHi (<i>B</i>), <i>M</i>. <i>catarrhalis</i> (<i>C</i>) or <i>P</i>. <i>aeruginosa</i> (<i>D</i>), and after extensive washing C5b-6 and C7 were added. After incubation for 10 min, C8 and C9 were added, and thereafter C5b-9 deposition on the microbial surface was detected with a mouse anti-C5b-9 mAb and Alexa 647-conjugated anti-mouse pAb by flow cytometry. E, vitronectin (50 μg/ml) was bound to immobilized proteins, and after extensive washing C5b-6 and C7 were added. After 10 min incubation, C8 and C9 were added, and C5b-9 deposition was detected with a mouse anti-C5b-9 mAb and an HRP-conjugated anti-mouse polyclonal antiserum. The mean values from three independent experiments are shown with error bars indicating SD. **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001.</p

Additional file 5: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Co-stimulation of exogenous CRAMP and bacterial supernatant NM induced increase of HO-1 immunofluorescence in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT or KO mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 6 h. After incubation cells were fixed and immunolabeled using anti-HO-1 antibody (green) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 3834 kb

Additional file 1: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Additional Material and Methods. Fluorescence microscopy of TUNEL and Ki67: Primary mice astrocytes or microglia were seeded on cover glasses. After stimulation for 24 h, the cells werr fixed with 4% paraformaldehyde. Subsequently, the cells were permeabilized with 0.1% Triton X in 0.1% sodium citrate for 3 min at 4°C. Then, the slices were incubated at 37°C for 1 h with TUNEL reaction mixture according the manufacturer’s protocol (In Situ Cell Death Detection Kit, Roche Diagnostics, Mannheim, Germany). After washing with PBS and blocking with 1.5 BSA in PBS for 10 min, the slices were incubated at 4°C about the night with Ki67 antibody (rabbit polyclonal; ab15580, abcam, UK). Finally, the slices were incubated with anti-rabbit Cy3 (AP132C, Millipore, Darmstadt, Germany) for 1 h at room temperature. Nuclear counter-staining was performed with Diamidino-2-phenylindole dihydrochloride DAPI (Sigma 9542, Munich, Germany). Cells were digitally photographed using a Keyence digital microscope (BZ-9000, Neu Isenburg, Germany). Ki67+ positive cells were counted for each treatment, where five 63×fields were evaluated. The proliferation index was determined by the number of positive cells expressing Ki67 divided by the total number of cells in each field. (DOCX 16 kb