Linoleic and palmitoleic acid block streptokinase-mediated plasminogen activation and reduce severity of invasive group A streptococcal infection

Abstract

In contrast to mild infections of Group A Streptococcus (GAS) invasive infections of GAS still pose a serious health hazard: GAS disseminates from sterile sites into the blood stream or deep tissues and causes sepsis or necrotizing fasciitis. In this case antibiotics do not provide an effective cure as the bacteria are capable to hide from them very quickly. Therefore, new remedies are urgently needed. Starting from a myxobacterial natural products screening campaign, we identified two fatty acids isolated from myxobacteria, linoleic and palmitoleic acid, specifically blocking streptokinase-mediated activation of plasminogen and thereby preventing streptococci from hijacking the host's plasminogen/plasmin system. This activity is not inherited by other fatty acids such as oleic acid and is not attributable to the killing of streptococci. Moreover, both fatty acids are superior in their inhibitory properties compared to two clinically used drugs (tranexamic or ε-amino caproic acid) as they show 500-1000 fold lower IC₅₀ values. Using a humanized plasminogen mouse model mimicking the clinical situation of a local GAS infection that becomes systemic, we demonstrate that these fatty acids ameliorate invasive GAS infection significantly. Consequently, linoleic and palmitoleic acid are possible new options to combat GAS invasive diseases.

Figure 1

Molecular structures of palmitoleic acid, linoleic acid, tranexamic acid and ε-amino caproic acid. The molecular structures of (a) palmitoleic acid (PA), (b) linoleic acid (LA), (c) tranexamic acid (TXA) and (d) ε-amino caproic acid (EACA) are displayed.

Figure 2

Inhibition of plasminogen activation in a cell-based plasminogen activation assay mediated by streptokinase cluster type 2a and 2b. LA and PA inhibit plasminogen activation mediated by streptokinase cluster type 2b (a) and 2a (c) in a concentration-dependent manner (micromolar range) in a cell-based plasminogen activation assay. By contrast, TXA and EACA only inhibit plasminogen activation by cluster type 2b (b) and 2a (d) at high concentrations (millimolar range). N = 3 for all experiments.

Figure 3

Inhibition of plasminogen activation in a supernatant-based plasminogen activation assay mediated by streptokinase cluster type 1 and 2a. LA and PA inhibit plasminogen activation mediated by streptokinase cluster type 2a (a) and 1 (c) in a concentration-dependent manner (micromolar range) in a supernatant-based plasminogen activation assay (a,c). By contrast, TXA and EACA only inhibit plasminogen activation mediated by streptokinase cluster type 2a (b) and 1 (d) at high concentrations (millimolar range) (b,d). N = 3 for all experiments.

Figure 4

LA and PA do not inhibit staphylokinase-mediated plasminogen activation, but inhibit plasminogen activation by recombinant streptokinase. (a) Plasminogen activation using staphylokinase. Compared to the untreated (w/o) control neither palmitoleic nor linoleic acid show an effect. (b) Plasminogen activation using recombinant cluster 2a type streptokinase. Both palmitoleic (PA) and linoleic acid (LA) (concentration for both: 40 µg/ml) inhibit plasminogen activation mediated by recombinant cluster 2a type streptokinase. w/o: untreated control. N = 3 for all experiments.

Figure 5

Prolongation of entrapment of streptococci within a clot upon treatment with LA and PA. Significantly fewer streptococci escape from a clot following treatment with LA or PA. (a) Streptococci harboring a cluster 2b type streptokinase were entrapped within a clot and treated with 60 µg/ml of either LA or PA or were left untreated. (b) Streptococci harboring a cluster 2a type streptokinase were entrapped within a clot and treated with 30 µg/ml of either LA or PA or were left untreated. cfu/ml in the supernatant were enumerated at 2 h and 4 h after treatment. *p < 0.05; **p < 0.01. N = 2 for all experiments.

Figure 6

Significant prolongation of survival in an invasive murine infection model using S. pyogenes strain AP1. LA and PA significantly prolong survival in an invasive murine infection model using S. pyogenes strain AP1 (p < 0.01). Transgenic humanized plasminogen mice were infected with 1 × 10⁷ cfu AP1 s.c., treated with 50 µg of either PA (n = 16) or LA (n = 16) or PBS s.c. (n = 15) (the experiment was performed four times with cohorts of 3–5 mice for each group).