Untreated mice received PBS subcutaneously (s

Untreated mice received PBS subcutaneously (s.c.) as a control on the day of infection. and palmitoleic acid are possible new options to combat GAS invasive diseases. Introduction Every year Leuprorelin Acetate millions of people suffer from group A streptococcal (GAS) diseases ranging from mild infections to severe and life-threatening syndromes including sepsis and necrotizing fasciitis. The latter are designated invasive diseases as bacteria are isolated from usually sterile sites such as deep tissues or the blood stream1. It is estimated that over 660,000 cases of invasive Group A (GAS) infections and over 160,000 deaths occur each year2. Even under treatment GAS invasive infections exhibit a high mortality rate of about 15C20%3. As a vaccine is not commercially available yet4,5, new drugs are urgently needed to successfully combat GAS invasive infections. GAS hijack the host factor plasminogen during invasive diseases6,7 by secreting streptokinase, a specific human plasminogen activator. Streptokinase activates plasminogen to plasmin, allowing GAS to disseminate Leuprorelin Acetate into deeper tissue8 or lyse fibrin Leuprorelin Acetate clots in which they may be entrapped9,10. Streptokinase is a single-chain, 414-amino-acid protein which is composed of three different domains: an -, – and a -domain11. Streptokinase can be classified into three so-called cluster types. Cluster 1 type streptokinase is secreted by streptococci and forms a complex with plasminogen directly, triggering a conformational change in the plasminogen molecule which then cleaves the Arg561-Val562 bond of another plasminogen molecule activating it to plasmin. Cluster 2 type streptokinase needs fibrinogen for activation of plasminogen. Cluster 2a type streptokinase is secreted and forms a tri-molecular complex with fibrinogen and plasminogen to activate plasminogen to plasmin. Cluster 2b type streptokinase is only able to activate plasminogen on the bacterial cell surface; plasminogen is bound to the streptococcal cell surface via plasminogen-binding group A streptococcal M or M-like protein. Then, a tri-molecular complex is formed (fibrinogen-plasminogen-streptokinase) activating further plasminogen molecules12,13. Additionally, it has been shown that cluster 2a type streptokinase can activate plasminogen in the absence of fibrinogen although it does not act as fast as cluster 1 type streptokinase14. Streptokinase can also form a complex with plasmin. This complex activates plasminogen more rapidly than a streptokinase-plasminogen-complex15. All three cluster types activate soluble plasminogen when formed into a streptokinase-plasmin-complex16. The 92?kDa single-chain plasminogen is a glycoprotein consisting of 791 amino acids17. A small molecule inhibitor directed against streptokinase has not been described. However, inhibitors of streptokinase gene expression have shown promise for the development of potential therapeutics18,19. Here, we identify two fatty acids isolated from myxobacteria, linoleic and palmitoleic acid, which block activation of plasminogen. Using a humanized plasminogen mouse model which mimics a local group A streptococcal infection that becomes systemic, we demonstrate that these fatty acids ameliorate invasive GAS infection. Thereby, Leuprorelin Acetate we provide evidence supporting the concept that these fatty acids can act as anti-virulence agents against GAS invasive infection. Consequently, linoleic and palmitoleic acid are possible new options for the treatment of invasive GAS disease. Results Natural products screening campaign reveals promising inhibitors of streptokinase-mediated plasminogen activation About 600 myxobacterial extracts and 300 myxobacterial compounds from our internal library were screened for their capacity of inhibition of the activation of plasminogen by streptokinase using well established assays to measure plasminogen activation by streptokinase13,14,16. Several myxobacterial extracts showed high inhibitory activity and reduced the generation of plasmin dramatically. To determine which peak in Rabbit polyclonal to PRKAA1 the chromatogram was responsible for activation, HPLC-fractionation was performed, revealing two peaks in the chromatogram responsible for the inhibitory activity in the plasminogen activation assay (Fig.?S1a,b). For isolation of the two compounds giving the activity in the chromatogram, the strain 70620 was selected as it yielded the highest inhibitory activity compared to equal amounts of other myxobacterial strains. To assure a high yield of both compounds, the strain 706 was optimized with respect to production of both compounds by testing different media and harvesting time points. The optimal harvesting time point and the optimal medium were selected due to the activity in the facilitated plasminogen activation assay. After fermentation of the strain 706 the compounds (RC 36.1 and RC 36.2) were isolated by activity-guided fractionation (Fig.?S1c,d) using different LC-MS fractionations that were tested in the facilitated plasminogen activation assay. Structure elucidation of RC 36.1 and RC 36.2 reveals them as palmitoleic and linoleic acid To elucidate the structure of.