The impact of Streptokinase on biofilm heterogeneity of group G streptococci

链激酶对G族链球菌生物膜异质性的影响

• 批准号: 503880638
• 负责人: Professor Dr. Michael Lalk
• 金额: --
• 依托单位: Arbeitskreis Stoffwechselbiochemie / Metabolomics
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/503880638?language=en
• 关键词: impact; Streptokinase; biofilm; heterogeneity; group

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.

停乳链球菌似马亚种（SDSE）是人类微生物群的主要组成部分，长期以来被认为比其他链球菌致病性低。然而，最近的研究报告了由SDSE引起的急性侵入性感染。目前，生物膜未被认为是链球菌感染的潜在问题，因为它通常与慢性感染有关或与外来器械有关。我们观察到“厚”生物膜社区在皮肤组织活检的几个患者感染SDSE和其他链球菌物种。此外，这些生物膜群落是异质多细胞的性质。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，是可渗透的细胞溶质探测，而第二个不是。我们的数据表明，第二个人口是多才多艺的性质。它可以保留在生物膜结构内或分散到周围组织中。SDSE分泌链激酶（Ska）。到目前为止，我们只知道Ska的一个功能。Ska结合人纤溶酶原，导致蛋白质复合物的构象变化。随后，蛋白质复合物溶解纤维蛋白凝块，允许SDSE在整个组织中扩散。我们观察到，形成多细胞生物膜群落的侵袭性SDSE的特征是Ska活性增强。此外，敲除Ska导致增强的生物膜形成。有趣的是，SDSEΔska的外源性Ska补充显著降低了生物膜质量。因此，我们的数据表明，一旦SDSE亚群形成稳定的生物膜群落，斯卡就会被释放。随后，Ska刺激群落内周围的细菌，这不允许它们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深层，也可以留在群落中。基于这些结果，我们假设，斯卡生物膜社区内的基本生物功能。它作为一种信号分子，刺激细菌的代谢特性。在这里，我们的目标是代谢这两个不同的人群和机械分析的信号斯卡。生物膜将生长，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、NMR和质谱法进行代谢组学分析。主要结果将使用组织工程方法进行验证。分析的代谢途径将提供候选途径和它们的潜在受体，这些受体是斯卡在多功能生物膜亚群中靶向的。基于这些分析，将进行候选基因的靶向诱变，并如上所述评价同基因突变体。