α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps - PubMed (original) (raw)

α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps

Yuka Mori et al. J Biol Chem. 2012.

Abstract

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia throughout the world, with high morbidity and mortality rates. A major feature of pneumococcal pneumonia is abundant neutrophil infiltration. In this study, we identified S. pneumoniae α-enolase as a neutrophil binding protein in ligand blot assay and mass spectrometry findings. Scanning electron microscopic and fluorescence microscopic analyses also revealed that S. pneumoniae α-enolase induces formation of neutrophil extracellular traps, which have been reported to bind and kill microbes. In addition, cytotoxic assay results showed that α-enolase dose-dependently increased the release of extracellular lactate dehydrogenase from human neutrophils as compared with untreated neutrophils. Furthermore, an in vitro cell migration assay using Chemotaxicell culture chambers demonstrated that α-enolase possesses neutrophil migrating activity. Interestingly, bactericidal assay findings showed that α-enolase increased neutrophil extracellular trap-dependent killing of S. pneumoniae in human blood. Moreover, pulldown assay and mass spectrometry results identified myoblast antigen 24.1D5 as an α-enolase-binding protein on human neutrophils, whereas flow cytometric analysis revealed that 24.1D5 was expressed on human neutrophils, but not on human monocytes or T cells. Together, our results indicate that α-enolase from S. pneumoniae increases neutrophil migrating activity and induces cell death of human neutrophils by releasing neutrophil extracellular traps. Furthermore, we found that myoblast antigen 24.1D5, which expressed on the surface of neutrophils, bound to α-enolase of S. pneumoniae.

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Figures

FIGURE 1.

Analysis of binding between neutrophil membrane-associated proteins and streptococcal surface proteins. Surface proteins from S. pneumoniae strain R6 were extracted with 8

urea, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were stained with Coomassie Brilliant Blue (CBB) or incubated with biotinylated erythrocyte membrane-associated proteins, followed by streptavidin-horseradish peroxidase. Interactions were detected with ECL Plus reagents. The band (arrow) demonstrated a reaction with biotinylated THP-1 cell surface proteins, which was identified by an MS/MS ion search.

FIGURE 2.

Localization of α-enolase on bacterial surface. A, urea extracts of S. pneumoniae strain R6 (Cell), cell culture supernatant (Sup), and purified recombinant α-enolase were analyzed by Western blotting using antiserum against α-enolase. Rabbit preimmune serum was used as a negative control. CBB, Coomassie Brilliant Blue. B, to confirm the localization of α-enolase on the surface of S. pneumoniae, bacterial cells were grown in anti-α-enolase serum and analyzed by immunofluorescence microscopy. The cells were washed with phosphate-buffered saline; then blocked with 1% Block Ace solution, 10% goat serum, and 5% BSA; and incubated with SYBR-GREEN I (green images) and rabbit anti-α-enolase serum (panels a and b) or preimmune serum as a negative control (panels c and d). Immunoreactive proteins were visualized with Alexa Fluor 594-conjugated goat anti-rabbit IgG (red images).

FIGURE 3.

Comparison of α-enolase in culture supernatants of S. pneumoniae wild-type and Δ_lytA_ strains. The amount of α-enolase in the culture supernatants was determined using ELISA, with that of the wild-type strain considered to be 100%. *, significant difference (p < 0.005) between the mean values, as determined with a Mann-Whitney U test. The S.E. values are represented by error bars (n = 6).

FIGURE 4.

Effects of α-enolase on neutrophil chemotaxis. Human neutrophils were isolated from whole blood samples, and their chemotactic reactions to α-enolase and C5a (10 n

; positive control) were examined. Chemotaxis is shown as fluorescence intensity. *, significant difference (p < 0.005) between the mean values, as determined with a Mann-Whitney U test. Three experiments were performed, with the data presented as the means of six wells from a representative experiment. The S.E. values are represented by error bars (n = 6).

FIGURE 5.

α-Enolase induces formation of NETs. A, scanning electron microscopic analysis of human neutrophils with or without α-enolase. B, quantitative enumerations of ruptured neutrophils per 20 fields of view. Euk-8 was used as a reactive oxidative species inhibitor. Three experiments were performed, and the data were presented from a representative experiment. C, cytotoxicity was determined using a lactate dehydrogenase assay. Maximal LDH release (100%) was obtained from cell lysates treated with Triton X-100, whereas the minimum release (0%) was from untreated cells. Three experiments were performed, with the data presented as the means of six wells from a representative experiment. The S.E. values are represented by error bars (n = 6). D, immunofluorescence analysis of human neutrophils with or without α-enolase. The cells were stained with SYTOX green (green) and Alexa Fluor 594 phalloidin (red). *, significant difference (p < 0.005) between the mean values, as determined with a Mann-Whitney U test.

FIGURE 6.

α-Enolase induces bacterial killing by NETs. S. pneumoniae (5 μl) cells were added to heparinized whole blood (85 μl) with or without DNase I (5 units, 5 μl) and α-enolase (final concentration, 10 n

, 5 μl) and then gently mixed for 1 h at 37 °C. Next, the mixture was serially diluted and plated on TS blood agar. Following incubation, the CFU values were determined. A, incubation with low doses (∼5 × 102 CFU) of S. pneumoniae strains R6 and D39. B, incubation with high doses of S. pneumoniae strains R6 and D39. *, significant difference (p < 0.005) between the mean values, as determined with a Mann-Whitney U test. Three experiments were performed, with the data presented as the means of six wells from a representative experiment. The S.E. values are represented by error bars (n = 6).

FIGURE 7.

Myoblast antigen 24.1D5 functions as α-enolase-binding protein. A, detection of α-enolase-binding protein by pulldown assay. THP-1 cell surface proteins were reacted with α-enolase on nickel-Sepharose. Proteins bound to α-enolase were separated by native PAGE, and then the gels were stained with a silver stain kit or transferred to polyvinylidene difluoride membranes. The membranes were incubated with anti-α-enolase serum, followed by goat anti-rabbit IgG. Interactions were detected using BCIP-NBT. The band (arrow) demonstrated an interaction with α-enolase, which was identified by an MS/MS ion search. B, analysis of binding between myoblast antigen 24.1D5 and α-enolase. Myoblast antigen 24.1D5 was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was then stained with Coomassie Brilliant Blue (CBB) or incubated with biotinylated α-enolase or BSA (negative control), followed by streptavidin-HRP. The interacted molecules were detected using ECL Plus reagents. C, the expression of myoblast antigen 24.1D5 was analyzed using a fluorescence-activated cell sorter system. The cells were prepared at 106 cells/ml and incubated with anti-myoblast antigen 24.1D5, the antibody as a positive control, and rabbit preimmune serum. The obtained data are presented as histograms.

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α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps - PubMed (original) (raw)