The impact of Streptokinase on biofilm heterogeneity of group G streptococci

欠乳链球菌（SDSE）亚种是人类微生物群的一种共生成分，长期以来被认为比其他链球菌的致病性低。然而，最近的研究报道了SDSE引起的急性侵袭性感染。目前，生物膜不被认为是链球菌感染的潜在问题，因为它通常与慢性感染或与外来装置有关。我们在几个感染SDSE和其他链球菌的患者的皮肤组织活检中观察到“厚”的生物膜群落。此外，这些生物膜群落具有异质多细胞性质。共聚焦激光扫描显微镜（CLSM）分析显示，SDSE生物膜由两个亚群组成。第一个群体，一个潜在的代谢不活跃，是渗透的细胞质探测，而第二个不是。我们的数据表明，第二种群在本质上是多才多艺的。它可以留在生物膜结构内或扩散到周围组织。SDSE分泌streptokinase （Ska）。到目前为止，人们只知道Ska的一个功能。Ska结合人纤溶酶原，导致蛋白复合物的构象改变。随后，蛋白质复合物溶解纤维蛋白凝块，使SDSE在组织中扩散。我们观察到，侵袭性SDSE形成多细胞生物膜群落，其特征是Ska活性增强。此外，敲除Ska导致生物膜形成增强。有趣的是，外源Ska补充SDSEΔska大大减少了生物膜质量。因此，我们的数据表明，一旦SDSE亚群形成稳定的生物膜群落，Ska就会被释放。随后，Ska刺激群落内周围的细菌，使其无法进入稳定的生物膜状态。这种代谢活跃的亚群可以扩散到组织的较深层，也可以留在群落中。基于这些结果，我们假设Ska在生物膜群落中具有基本的生物学功能。它作为一种信号分子，刺激细菌的代谢特性。在这里，我们的目标是代谢分析这两个不同的群体和机制分析Ska信号。生物膜将被培养，不同的群体将被荧光标记，其蛋白质生化，代谢和机械特性将被分析。此外，生物膜群落将根据其代谢活性进行分类，随后使用成像，核磁共振和质谱方法进行代谢组分析。主要结果将使用组织工程方法进行验证。分析代谢途径将提供Ska在多功能生物膜亚群落中靶向的候选途径及其潜在受体。基于这些分析，将对候选基因进行靶向诱变，并对等基因突变体进行如上所述的评估。

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.