Escaping the Salmonella-containing vacuole – Role of flagella-mediated damage, cytosolic motility, and host cell apoptosis in exit and transmission of Salmonella

该项目研究鞭毛和细胞内运动的作用，从宿主细胞的出口。我们先前观察到，含沙门氏菌的空泡（SCV）的完整性对于细胞内沙门氏菌和宿主细胞的命运是决定性的，并且SCV的损伤可导致细菌过度复制、细胞从上皮层驱逐或诱导凋亡性细胞死亡。产生的凋亡小体可能窝藏并将活沙门氏菌传播到幼稚宿主细胞。我们最近观察到，与S.肠血清型鼠伤寒（STM）、伤寒血清型甲型副伤寒（SPA）在逃离SCV后表达功能性鞭毛，并且是细胞内移动的。我们的目的是了解特定的细胞内生活方式的STM和SPA，SCV损伤的机制和沙门氏菌的胞质活动的后果。特别是，我们将调查： ·细胞内鞭毛表达的动力学，理解SPA与STM中的调节·沙门氏菌鞭毛在SCV损伤、诱导宿主细胞应答和宿主细胞退出中的作用·鞭毛对新生SCV的损伤机制和鞭毛在入侵期间的命运我们将部署活细胞报告基因EqtSM来监测SCV损伤并分析细菌和宿主细胞因素对SCV损伤或稳定性的贡献：·应用EqtSM报告细胞系以高空间和时间分辨率分析SCV损伤·建立用于分析SPA细胞内生活方式的改进的类器官模型系统·产生控制感染宿主细胞中鞭毛功能的光遗传学实验系统·使用感染宿主细胞的活细胞成像和巨大单层囊泡中捕获的沙门氏菌的最小系统分析对空泡损伤的贡献我们将研究沙门氏菌如何通过携带活细菌的凋亡小体感染宿主并解决以下问题：宿主细胞的热凋亡性细胞死亡和通过凋亡小体释放沙门氏菌是感染幼稚宿主细胞的受保护途径吗？·AB中STM的细胞内环境是什么，以及宿主细胞吞噬后的AB？预期的结果将扩大我们对沙门氏菌感染中宿主病原体相互作用、伤寒沙门氏菌感染的发病机制和传播途径的理解。

The project investigates the role of flagella and intracellular motility for the exit from the host cell. We previously observed that integrity of the Salmonella-containing vacuole (SCV) is decisive for the fate of intracellular Salmonella and the host cells, and damage of the SCV can lead to bacterial hyper-replication, expulsion of cells from epithelial layers, or induction of apoptotic cell death. Resulting apoptotic bodies may harbour and transmit viable Salmonella to naïve host cells. We recently observed that in contrast to S. enterica serovar Typhimurium (STM), the typhoidal serovar Paratyphi A (SPA) expressed functional flagella after escape from the SCV and is intracellular mobile. We aim to understand the specific intracellular lifestyles of STM and SPA, mechanisms of SCV damage and consequence of cytosolic activities of Salmonella. In particular, we will investigate: • Kinetics of intracellular flagella expression, understanding regulation in SPA vs. STM • Role of Salmonella flagella in SCV damage, induction of host cell responses, and host cell exit • Mechanics of flagella damage to nascent SCV and fate of flagella during invasion We will deploy the live cell reporter EqtSM to monitor SCV damage and to analyse the bacterial and host cell factors contribution to SCV damage or stability: • Application of EqtSM reporter cell lines to analyse SCV damage with high spatial and temporal resolution • Further investigation of Salmonella factors (T3SS effector proteins) involved in SCV damage and stabilization • Set up of improved organoid model systems for analyses of SPA intracellular lifestyle • Generation of optogenetic experimental systems to control flagella functions in infected host cells • Analyses of contribution to vacuolar damage using live cell imaging of infected host cells and minimal system of Salmonella entrapped in giant uni-lamellar vesicles We will investigate how Salmonella can infect host via apoptotic bodies harbouring viable bacteria and address the questions: • Is host cell pyroptotic cell death and release of Salmonella by apoptotic bodies a protected route for infection of naïve host cells? • What is the intracellular environment of STM in AB, and AB after phagocytosis by host cells? The anticipated results will extend our understanding of host pathogen interaction in Salmonella infections, the pathogenesis of typhoidal Salmonella infections, and routes of transmission.