The impact of Streptokinase on biofilm heterogeneity of group G streptococci

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Streptococcus dysgalactiae subspecies equisimilis (SDSE) is a commensal constituent of the human microbiota and was long considered less pathogenic than other streptococci. However, recent studies have reported acute invasive infections caused by SDSE. At present, biofilm is not recognized as a potential problem in streptococcal infections, as it is typically linked to chronic infections or associated with foreign devices. We observed “thick” biofilm communities in the skin tissue biopsies of several patients infected with SDSE and other streptococcal species. Moreover, these biofilm communities are of heterogenic multicellular nature. Confocal laser scanning microscopy (CLSM) analyses revealed that SDSE biofilms consist of two subpopulations. First population, a potentially metabolically inactive, is permeable for cytosolic probing, while the second is not. Our data indicate that the second population is versatile in nature. It can remain within the biofilm structure or disperses to the surrounding tissue. SDSE secrete streptokinase (Ska). So far, only one function of Ska is known. Ska binds human plasminogen resulting in conformational change of the protein complex. Subsequently, the protein complex lyses fibrin clots allowing SDSE to spread across the tissue. We observed that invasive SDSE, which form multicellular biofilm community, are characterized by enhanced Ska activity. Furthermore, knock-out of Ska results in in enhanced biofilm formation. Interestingly, exogenous Ska supplementation of SDSEΔska substantially reduces biofilm mass. Therefore, our data indicates that once a subpopulation of SDSE forms a stable biofilm community, Ska gets released. Subsequently, Ska stimulates the surrounding bacteria within the community, which does not allow them to enter a stable biofilm state. This metabolically active subpopulation can either spread to the deeper layers of the tissue or remain within the community. Based on these results, we hypothesize that Ska has fundamental biological function within the biofilm community. It functions as a signal molecule, which stimulates bacterial metabolic properties. Here, we aim to metabolically profile these two distinct populations and mechanistically analyze the signaling of Ska. Biofilms will be grown, the distinct populations will be fluorescently labeled and its protein-biochemical, metabolic, and mechanical properties will be analyzed. Furthermore, biofilm communities will be sorted based on their metabolic activity and subsequently subjected to metabolome analysis using imaging, NMR and mass spectrometry methods. Major results will be validated using tissue engineering approaches. The metabolic pathways analyzed will provide candidate pathways and their potential receptors that are targeted by Ska within the versatile biofilm subcommunity. Based on these analyses, targeted mutagenesis of candidate genes will be performed and the isogenic mutants will be evaluated as described above.