Fig 2: PSMa4-induced HBP release increases HVUEC monolayer permeability.(A) PSMa4 did not cause cytotoxicity in HUVECs. HUVECs (104/well) were seeded in 24-well plates and incubated for 24 h. The HUVECs were then incubated with PSMa4 at 10, 20, or 30 µg/mL at 37 °C for 30 min. The supernatant was collected and cytotoxicity was analyzed by LDH assay. (B) PSMa4-induced HBP release increased HVUEC monolayer permeability. The HUVEC monolayer on the transwell insert chamber was incubated with d-mannitol (1.4 mM), culture supernatant of whole blood treated with PSMa4 (10 µg/mL), the supernatant + functional blocking antibody against HBP, the supernatant + the isotype control IgG (50 µg/mL), PSMa4 (10 µg/mL), or PBS at 37 °C for 30 min. Lucifer yellow was also added to the incubation media. The fluorescence intensity in the lower chamber of the transwells was determined. d-mannitol and PBS were used as the positive and negative control, respectively.

Fig 3: PSMa4 stimulates HBP release from PMNs via FPR2 and PI3K signaling pathway.(A) FPR2 antagonist WRW4 significantly reduced HBP release from whole blood. Whole blood was pre-incubated with WRW4 (50 µg/mL) or the scramble all d-amino acid control wwrw3 (50 µg/mL) at 37 °C for 15 min30 and then treated with PSMa4 (10 µg/mL) at 37 °C for 30 min. HBP in the supernatant was analyzed by ELISA. (B) Formyl PSMa4 peptide induced HBP release dose-dependently. Human whole blood was incubated with 10, 20, or 30 µg/mL non-formyl or formyl PSMa4 at 37 °C for 30 min. PBS was used as the negative control. (C) The critical amino acid residues of PSMa4 to induce HBP release were screened by alanine substitution. The concentration of the peptides was 10 µg/mL. Replacement of I3, V4, G5, T6, I11, I15, I17, or F18 with alanine significantly abolished HBP release. (D) PI3K-specific inhibitor wortmannin completely abolished HBP release. (E) Rac-specific inhibitor NSC23766 completely abolished HBP release. Whole blood was pre-incubated with 1 µM wortmannin or 50 µM NSC23766 at 37 °C for 1 h and then treated with PSMa4 (10 µg/mL) for 30 min. HBP in the supernatant was analyzed by ELISA. (F) EGTA in culture media significantly reduced HBP release. Human whole blood was pre-treated with 20 mM EGTA at 37 °C for 15 min and then stimulated with PSMa4 (10 µg/mL). (G) PSMa4 induced Ca2+ influx into PMNs. Fluo-3/AM was loaded to PMNs before stimulation with PSMa4. fMLP (1 µM) was used as a positive control for Ca2+ influx.

Fig 4: PSMa4 from S. aureus culture supernatant stimulates HBP release from whole blood of healthy donors.(A) Blood specimens from patients infected with S. aureus contained significantly higher levels of HBP than blood from healthy donors. (B) USA300 supernatant induced HBP release from whole blood in a dose-dependent manner (10%, 20%, and 30%). fMLP (100 nM) + cytochalasin B (5 µg/mL), LTB4 (20 ng/mL) + cytochalasin B (5 µg/mL), and SLO (10 µg/mL) were used as positive controls for HBP release. PBS was used as the negative control. (C) Fractions 8 to 14 eluted from a Resource Q column induced HBP release. (D) High-resolution SDS-PAGE revealed a unique peptide component (3 kDa) in the eluted fractions stimulating HBP release. (E) The small peptide was soluble in ethanol. (F) Ethanol-soluble peptides stimulated HBP release from whole blood in a dose-dependent manner, while ethanol-insoluble peptides weakly induced HBP release. (G) Supernatant from the PSMa deletion mutant (?a) failed to induce HBP release. Supernatant from the complemented ?a strain (a4comp?a) showed normal HBP release. (H) Synthetic PSMa4 peptide stimulated HBP release in a dose-dependent manner while PSMa1–3 did not stimulate HBP release from whole blood. (I) Culture supernatant of CA-MRSA stimulated HBP release to a higher extent than that of HA-MRSA. CA-MRSA strain numbers: 1, 87, 129, 130, and 131. HA-MRSA strain numbers: 70, 107, 108, 120, and 206. ***P < 0.001, **P < 0.01, *P < 0.05.