Cytolysin-mediated neutrophil activation in streptococcal infections – mode of action and consequences for the human host

中性粒细胞是血液中的主要白细胞成分，也是细菌感染部位的第一批应答者。描述了三种不同的抗感染机制：吞噬、脱颗粒和中性粒细胞胞外陷阱(Net)的形成。所有这些过程的关键成分都是颗粒效应分子，包括蛋白水解酶和抗菌肽。链球菌包括化脓性链球菌、乳链球菌亚种。等同菌株和肺炎链球菌进化出了一套截然不同的蛋白质细胞毒素，干扰了中性粒细胞的重要功能。其中之一是它们的溶细胞性。它们与富含胆固醇的膜相互作用，导致寡聚、孔洞形成，以及随后由中性粒细胞成分介导的组织病理。然而，我们的数据和来自其他实验室的数据表明，这些毒素也可以在亚溶解浓度下激活中性粒细胞，而不需要溶解。在这项建议中，我们的目标是分析链球菌溶血素O和肺炎溶血素的中性粒细胞激活特性，具体地说，我们的目标是：(I)定义中性粒细胞激活所需的各自毒素的最低量，(Ii)确定潜在的中性粒细胞受体是毒素的靶标，(Iii)描述相互作用和由此产生的后果，以及(Iv)确定能够防止中性粒细胞过度激活和随后的组织病理的天然和治疗性中和剂。我们将对感染和/或刺激的人类初级中性粒细胞进行全面的微生物、免疫学和生化分析。此外，我们还将在患者S的材料中验证所获得的结果。

Neutrophils are the major leukocyte component of the blood and the first recruited responders at the site of bacterial infection. Three distinct mechanisms to fight an infection are described: phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs). Critical components of all these processes are granule effector molecules, including proteolytic enzymes and antimicrobial peptides. Streptococcal species, including S. pyogenes, S. dysgalactiae subsp. equisimilis, and S. pneumoniae have evolved a distinct repertoire of protein cytotoxins that interfere with crucial neutrophil functions. One of them is their cytolytic property. Their interactions with cholesterol-rich membranes result in oligomerization, pore formation, and subsequent tissue pathology mediated by neutrophil components. However, our data and data from other laboratories suggest that these toxins can also activate neutrophils at sub-lytic concentrations without lysis. In this proposal, we are aiming to analyze neutrophil activating properties of streptolysin O and pneumolysin, Specifically, we aim to: (i) define the minimum amount of the respective toxin which is necessary for neutrophil activation, (ii) identify potential human neutrophil receptors which are targeted by the toxins, (iii) characterize the interaction and the resulting consequences, and (iv) identify natural and therapeutic neutralizing agents to be able to prevent excessive neutrophil activation and subsequent tissue pathology. We will perform comprehensive microbiological, immunological, and biochemical analyses of infected and/or stimulated human primary neutrophils. Furthermore, we will verify the obtained results in patient´s material.