Staphylococcus aureus induces release of bradykinin in human plasma

Abstract

Staphylococcus aureus is a prominent human pathogen. Here we report that intact S. aureus bacteria activate the contact system in human plasma in vitro, resulting in a massive release of the potent proinflammatory and vasoactive peptide bradykinin. In contrast, no such effect was recorded with Streptococcus pneumoniae. In the activation of the contact system, blood coagulation factor XII and plasma kallikrein play central roles, and a specific inhibitor of these serine proteinases inhibited the release of bradykinin by S. aureus in human plasma. Furthermore, fragments of the cofactor H-kininogen of the contact system efficiently blocked bradykinin release. The results suggest that activation of the contact system at the surface of S. aureus and the subsequent release of bradykinin could contribute to the hypovolemic hypotension seen in patients with severe S. aureus sepsis. The data also suggest that the contact system could be used as a target in the treatment of S. aureus infections.

FIG. 1

BK levels in human plasma samples. Samples 1 to 4 are from patients with S. aureus sepsis, 5 to 10 are from patients with S. pneumoniae sepsis, and 11 to 13 are from healthy controls. The BK levels were determined with an ELISA. Values are means of two different determinations. The variation was <15%.

FIG. 2

Cleavage of HK at the surface of S. aureus. S. aureus (10¹⁰ CFU/ml) was incubated with human plasma (final concentration, 50%) for 15 min. Subsequently, bacteria were washed, resuspended in buffer, incubated for another 15 min, and spun down. The resulting supernatant and plasma (nontreated or kaolin treated) were then subjected to SDS-PAGE (10% polyacrylamide gel), and the separated proteins were transferred to Immobilon filters and probed with antibodies to HK. Lanes: 1, normal plasma; 2, kaolin-treated plasma; 3, proteins absorbed from plasma by S. aureus.

FIG. 3

S. aureus induces BK release in human plasma. Various numbers of S. aureus or S. pneumoniae bacteria were incubated with human plasma (final concentration of 50%) for 15 min. Bacteria were washed and resuspended in buffer. Following another incubation period of 15 min, bacteria were pelleted, and the BK content of the resulting supernatants was determined with an ELISA. Plasma alone was the negative control. Values are means ± standard deviations (n = 3).

FIG. 4

Kinetics of BK release induced by S. aureus in human plasma. (A) Bacteria were incubated (10¹⁰ CFU/ml) with 50% human plasma for different time periods. After washing, bacteria were resuspended in buffer and incubated for 15 min. (B) Bacteria (10¹⁰ CFU/ml) were incubated with 50% plasma for 15 min, washed, resuspended in buffer, and incubated for different time periods. In both panels A and B, bacteria were spun down and the BK content in supernatants was measured with an ELISA. Values are means ± standard deviations (n = 3).

FIG. 5

Incubation of S. aureus in plasma induces the release of functional BK. (A) Different amounts of BK were added to CHO-K1 cells, and the release of ³H-labeled inositol phosphate was expressed as disintegrations per minute (DPM). (B) Samples from preincubations of plasma with buffer (1:1 [background]) or from preincubations of the indicated numbers of bacteria with plasma (final concentration of 50%) were added to CHO-K1 cells in the presence or absence of the BK B2 receptor inhibitor HOE 140 (8 μM). The release of inositol phosphate was measured. Values are means ± standard deviations (n = 3).

FIG. 6

Inhibition of S. aureus-induced BK release. S. aureus bacteria (10¹⁰ CFU/ml) were incubated for 15 min with plasma in the absence (100% of control) or presence of a peptide inhibitor of FXII-PK (HD-Pro-Phe-Arg-CMK), domain D5 of HK, or peptides derived from D5 (HKH20 and GHG19). E64, a specific cysteine proteinase inhibitor, served as a negative control. After washing and incubation with buffer (15 min), the bacteria were spun down, and the BK content in the supernatant was determined. Values are means ± standard deviations (n = 3).