Activation of TAFI on the surface of Streptococcus pyogenes evokes inflammatory reactions by modulating the kallikrein/kinin system

Abstract

Bacteria-controlled regulation of host responses to infection is an important virulence mechanism that has been demonstrated to contribute to disease progression. Here we report that the human pathogen Streptococcus pyogenes employs the procarboxypeptidase TAFI (thrombin-activatable fibrinolysis inhibitor) to modulate the kallikrein/kinin system. To this end, bacteria initiate a chain of events starting with the recruitment and activation of TAFI. This is followed by the assembly and induction of the contact system at the streptococcal surface, eventually triggering the release of bradykinin (BK). BK is then carboxyterminally truncated by activated TAFI, which converts the peptide from a kinin B(2) receptor ligand to a kinin B(1) receptor (B1R) agonist. Finally, we show that streptococcal supernatants indirectly amplify the B1R response as they act on peripheral blood mononuclear cells to secrete inflammatory cytokines that in turn stimulate upregulation of the B1R on human fibroblasts. Taken together our findings implicate a critical and novel role for streptococci-bound TAFI, as it processes BK to a B1R agonist at the bacterial surface and thereby may redirect inflammation from a transient to a chronic state.

Fig. 1

Processing of HK and release of BK at the bacterial surface. A S. pyogenes bacteria of serotype M41 were incubated with human plasma for 60 min followed by a washing step to remove unbound proteins. Bacteria-absorbed proteins were eluted with an acid wash and the recovered proteins were subjected to Western blot analysis and immunodetection with antibodies against HK. Lane 1 = Normal human plasma; lane 2 = kaolin-treated human plasma, which leads to a complete cleavage of HK into its heavy and light chain; lane 3 = proteins absorbed from plasma by S. pyogenes. B Bacteria were incubated with medium or plasma for 15 min followed by a washing step to remove unbound proteins. Bacteria were then resuspended in buffer for another 15 min to allow activation of the contact system and the release of BK into the liquid phase. Supernatants were collected and BK concentrations were determined by ELISA. The BK content in the plasma samples used for the experiments was measured and considered as background. Values are means ± standard deviations (n = 3). * p < 0.01 by Student's t test.

Fig. 2

Detection of BK and desArg9BK by HPLC. Commercially available BK (1 µM; A) and desArg⁹BK (1 µM; B) were analyzed by HPLC. BK (1 µM) was incubated with plasmin-activated TAFI (20 nM) for 15 min followed by HPLC analysis of BK cleavage products (C). AP41 bacteria (2 × 10⁹ cells/ml) were incubated with (10 µg/ml) plasmin for 60 min on ice, followed by a washing step to remove unbound protein (D). This was followed by the addition of TAFI (20 nM) and after another 20-min incubation at room temperature, BK was added to the mixture and incubated for 15 min at 37°C. Bacteria were removed by a centrifugation step and the supernatants were analyzed by HPLC. The HPLC chromatograms are representative of at least four separate experiments.

Fig. 3

Transmission electron micrograph of gold-labeled TAFI with gold-labeled BK and desArg⁹BK. TAFI was labeled with 40 nm colloidal gold and incubated with 15 nm gold-labeled BK (A) or desArg⁹BK (B). Samples were prepared for electron microscopy by negative staining with uranyl formate. Low magnification fields of complexes are shown. Arrowheads point to gold-labeled kinins that are in complex with gold-labeled TAFI. Statistical evaluation revealed that 49% of gold-labeled TAFI were in complex with gold-labeled BK, while only 8% were associated with gold-labeled desArg⁹BK (C). Representative complexes between gold-labeled TAFI and gold-labeled BK are depicted in the inserts. The bar represents 200 (A, B) and 25 nm (C).

Fig. 4

Transmission electron micrograph of gold-labeled TAFI with gold-labeled BK at the surface of AP41 bacteria. A AP41 bacteria (2 × 10⁹ cells/ml) were incubated with (10 µg/ml) plasmin for 60 min on ice, followed by a washing step to remove unbound protein. This was followed by adding gold-labeled TAFI and after another 20 min gold-labeled BK was added to the mixture. Bacteria were pelleted and then subjected to negative staining electron microscopy. Arrows indicate gold-labeled TAFI in complexes with gold-labeled BK attached to the bacterial surface. B Representative complexes between gold-labeled TAFI and gold-labeled BK are depicted in the inserts. The bar represents 100 (A) and 50 nm (B).

Fig. 5

Detection by HPLC of BK and desArg⁹BK released from AP41 bacteria. A AP41 bacteria (2 × 10⁹ cells/ml) were incubated with (10 µg/ml) plasmin for 60 min on ice, followed by a washing step to remove unbound protein. Afterwards (20 nM) TAFI-IIYQ was added for 20 min and after a second washing step BK was given to the mixture and incubated for 15 min at 37°C. Bacteria were removed by a centrifugation step and the supernatants were analyzed by HPLC. B AP41 bacteria were first treated with TAFI-IIYQ and then with plasmin. Washings steps and incubation with BK were performed as described in A. The HPLC chromatograms are representative of at least four separate experiments.

Fig. 6

Measurements of the biological activity of desArg⁹BK released from AP41 bacteria and cell surface expression of B1R. A HEK293 cells stably transfected with B1R were treated with (i) buffer, (ii) the B1R antagonist DLKD, (iii) desArg⁹BK, (iv) a mixture of desArg⁹BK and DLKD, (v) BK cleavage products released from AP41 bacteria, and (vi) a mixture of BK cleavage products released from AP41 bacteria and DLKD. Inositol phosphates were detected as described in Materials and Methods. The result is representative of three experiments with each point performed in duplicates. B IMR-90 cells were incubated for 6 h with (i) medium, (ii) PBMC exudates (monocytic cells that had been stimulated for 24 h with 1% S. pyogenes overnight culture supernatants), or (iii) PBMC exudates in the presence of 10 µM desArg⁹BK. All treatments were performed in MEM medium and in the absence of serum and antibiotics. After intensive washing, cells were assayed for specific [³H]des-Arg¹⁰kallidin (B1R ligand) binding. Binding of [³H]des-Arg¹⁰kallidin to non-stimulated cells (control) was normalized to 100% within each experiment. Results represent the mean ± standard deviation of 3 independent experiments performed in triplicates. * p < 0.01 by Student's t test.

Fig. 7

Proposed mechanism used by S. pyogenes (AP41) to modulate the inflammatory response. Based on the present study, the following model is suggested. Plasma exudation into the infectious site will allow S. pyogenes bacteria to bind human TAFI via SclA and SclB. TAFI activators such as plasmin and thrombin/thrombomodulin can also be recruited to the bacteria and trigger activation of bound TAFI. At the same time bacteria assemble and activate the factors of the contact system at their surface which results in the generation of BK. Invading monocytes at the infected site become activated by streptococcal secretion products, i.e. exotoxins, and evoke the secretion of proinflammatory cytokines that in turn trigger an upregulation of B1R at the infectious focus. The upregulated B1R is now accessible for desArg⁹BK, released from the bacteria, which may contribute to a sustained inflammatory response.