Group A Streptococcus remains viable inside fibrin clots and gains access to human plasminogen for subsequent fibrinolysis and dissemination

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes a wide spectrum of illnesses ranging from pharyngitis and rheumatic fever to more invasive and severe diseases such as necrotizing fasciitis and toxic shock syndrome. Invasive outcomes of GAS infections often result from entry of the bacteria via an open wound into tissue and blood systems. The coagulation cascade serves as an innate defense mechanism that initiates fibrin clots to sequester bacteria and restrict its growth and prevent dissemination into deeper tissues. GAS, especially skin-tropic bacterial strains, utilize the specific virulence factors Plasminogen binding M-protein (PAM) and streptokinase (SK) to manipulate hemostasis and ultimately activate human plasminogen to cause fibrinolysis and escape from the fibrin clot. A major unresolved question regarding this process is to understand the temporal dynamics of how GAS that is enmeshed in a fibrin clot accesses host plasminogen for dissolution of the clot and eventual dissemination. Using fluorescently labeled plasminogen and fibrinogen, we established conditions to observe the process of fibrin clot dissolution by GAS (an AP53 CovR+S-strain) that is sequestered in a fibrin clot using real-time imaging microscopy. We hypothesized that initiation of fibrinolysis by GAS inside a fibrin clot would be determined by the rate of hPg access into the fibrin clot where bacteria are trapped. Our live imaging studies show that GAS trapped inside a fibrin clot, has limited access to hPg; however, at 4.25 h post incubation, when sufficient hPg is accessible to the bacterium, fibrinolysis quickly occurs. If hPg is bound to the bacterial surface prior to being trapped inside a clot, dissolution and bacterial dissemination occurs at a much faster rate of 2.5 h post incubation. During the time which bacteria are trapped in the clot without access to hPg, we did not observe any growth of GAS; however, we demonstrate that the bacteria continue to remain viable inside the fibrin clot. We performed RNA-seq analysis of GAS and the isogenic GAS SK-deficient mutant to understand SK-dependent transcriptional changes during the lag-phase of the GAS bacteria inside the fibrin clot. We observed a dramatic change in the transcription profile of wt GAS inside the fibrin clot over time prior to escape from the fibrin clot (22 gene expression changes at 4h, to 802 gene expression changes at 8h). Furthermore, we also identified gene expression changes that were distinct between wt GAS and the GAS SK-deficient mutant. Our findings reveal for the first time that GAS can engage a latent, growth suspended phase whereby physical structures such as fibrin clots and Neutrophil extracellular traps that immobilize an invading pathogen allow bacteria to remain viable and transcriptionally active for an extended time during host infection. GAS that is trapped in a fibrin clot will therefore enter a state in which the bacteria suspend growth, but remain viable, until sufficient access to hPg allow it to initiate fibrinolysis and escape into surrounding tissues. The viability of GAS while trapped and its readiness to avoid immune defenses allow GAS to act quickly to disseminate when host conditions are more favorable for the bacteria. ### Competing Interest Statement The authors have declared no competing interest.