Figure 1.
StcE binds to the neutrophil surface and interacts with CD43 and CD45.
(A) Neutrophils were incubated with vehicle control, purified StcE protein, or the proteolytically inactive mutant E435D at 1 µg/mL. Cells were stained with polyclonal anti-StcE and analyzed by flow cytometry for StcE binding. (B) StcE fused to a chitin binding domain (StcE-CBD) was bound to chitin beads (CB) and used to precipitate binding partners. dHL-60 cells (5×106) were lysed and incubated with StcE-CBD bound to CB or CB alone. Pulldown reactions were separated by SDS-PAGE and immunoblotted for CD43 (L10) or CD45 (HI-30).
Figure 2.
StcE cleaves within the extracellular domain of neutrophil CD43 and CD45.
(A) Intact dHL-60s (1×106) were treated with 1 µg/mL purified, endotoxin-free StcE, E435D or vehicle control. Supernatants and cell pellets were individually separated by SDS-PAGE and immunoblotted with L10, which recognizes an extracellular epitope near the N-terminus of CD43. (B) Reactions were performed as in A) and immunoblotted with sc-7052, which recognizes the intracellular C-terminus of CD43. (C) Primary human neutrophils were treated as in (A) and analyzed by flow cytometry to detect the CD43 extracellular domain using PE-labeled L10. (D) Reactions were performed as described in (A) and immunoblotted with HI30, which recognizes all CD45 isoforms. (E) Reactions were performed as in (C) and detected with HI30. F) Reactions were performed as in (C) and detected with UCHL1, which recognizes only the CD45RO isoform.
Figure 3.
Proteolytic activity of StcE is required to induce the oxidative burst, but not neutrophil adhesion.
(A and B) Neutrophils were labeled with dihydrorhodamine 123, treatmed with 5 µg/mL StcE, E435D, or vehicle control, and analyzed by flow cytometry for oxidative burst production as indicated by fluorescence in the FITC channel. PMA served as a positive control. The live cell population was gated by forward and side scatter, and then the “percent positive” gate was drawn to distinguish PBS-treated (negative) and PMA-treated (positive) samples. (A) shows data from a representative experiment, while B) shows the average of five independent experiments. Error bars represent S.D.; *, p<0.05; **, p<0.01 by one-way ANOVA with Bonferroni post test. (C) Neutrophils were fluorescently labeled with calcein-AM, treated with vehicle control, StcE, or E435D at varying concentrations, and allowed to adhere on Fbg for 40 min. Positive control samples were stimulated with 100 nM fMLP for the final 10 min (dotted line). Relative adhesion was calculated by normalizing the number of adherent cells to the average of all vehicle-treated samples. Data shown are mean±S.E.M. of three independent experiments performed in duplicate. *, p<0.05; **, p<0.01; ***, p<0.001 compared to equivalent vehicle control using two-way ANOVA with Bonferroni post test.
Figure 4.
Relocalization of CD43 by the E435D mutant.
(A) Neutrophils were treated with 20 µg/mL StcE or E435D for 30 min, followed by 10 min of treatment with 100 nM fMLP to induce firm adhesion and polarization. Cells were fixed and stained with rabbit anti-StcE and mouse anti-CD43 L10 and analyzed by confocal immunofluorescence microscopy. (B) Neutrophils were treated as in (A) and stained for the CD43 intracellular domain with sc-7052. Scale bar represents 10 µm for all images.
Figure 5.
StcE inhibits neutrophil chemotaxis.
(A) Neutrophils labeled as in figure 3C were treated with 25 µg/mL StcE or E435D in the upper chamber of Fbg-coated transwell inserts, and media with or without chemoattractant (100 nM fMLP) placed in the lower chamber. Cells were allowed to migrate for 210 min and neutrophils in the lower chamber were quantified and normalized as described. Data shown are mean±S.E.M. of three independent experiments. **, p<0.01 compared to vehicle with chemoattractant by one-way ANOVA using Dunnett post test. (B) Experiments were performed as described in (A), with 50 µg/mL protein and monolayers of HMVEC-L grown on collagen-coated transwell inserts. Data are mean±S.E.M. from six independent experiments analyzed as in (A).
Figure 6.
StcE decreases neutrophil random migration via impaired uropod retraction.
Neutrophils were treated with human serum albumin (HSA), StcE, or E435D (12.5 µg/ml) in Fbg-coated dishes for 30 min and stimulated with IL-8 for the final 5 min where indicated. Neutrophil migration was imaged by time lapse video microscopy over 10 min. Still images were captured and compiled into videos using Metamorph software (see Videos S1, S2, S3, S4, S5, and S6), and image tracking was performed. (A) Still images from a representative experiment are shown with scale bar representing 10 µm. Graphs of cell tracks below still images indicate individual paths of all cells tracked for each condition over 10 min. (B) Cell velocity was calculated using MetaMorph software for IL-8 stimulated samples. Data are presented as mean±S.E.M. of four independent experiments. *, p<0.05 as compared to HSA by one-way ANOVA with Dunnett's post test. (C) Quantitation of cell length in stimulated and unstimulated samples was performed using Metamorph by measuring the length of all cells in two randomly chosen frames per sample. Data are shown as mean±S.E.M of three independent experiments; *, p<0.05 compared to appropriate HSA control by two-way ANOVA with Bonferroni's post test.
Figure 7.
StcE induces mislocalization of zebrafish neutrophils.
(A) Zebrafish embryos at 3 dpf were wounded in the ventral fin in the presence of 25 µg/mL StcE, hiStcE, or vehicle control and fixed after 24 hours for whole-mount immunofluorescence with anti-mpo staining. Arrows indicate abnormal localization of neutrophils in the fin of StcE-treated zebrafish. The edge of the fin is outlined in white, and the site of the wound is indicated by an asterisk. The scale bar represents 200 µm. (B) Images from independent experiments were blinded and the number of neutrophils in the fin counted. Data are shown as means of individual experiments (shapes)±S.E.M. and total combined mean (bars); **, p<0.01 of combined means by one-way ANOVA with Dunnett's post test.