Function of Legionella pneumophila phospholipases in vacuole and host egress

嗜肺军团菌磷脂酶在液泡和宿主出口中的功能

• 批准号: 446411117
• 负责人: Professorin Dr. Antje Flieger
• 金额: --
• 依托单位: Fachgebiet 11: Bakterielle darmpathogene Erreger und Legionellen
• 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/446411117?language=en
• 关键词: Function; Legionella; pneumophila; phospholipases; vacuole

Legionella pneumophila (Lpn), an important intracellular bacterial pathogen, infects the human lung and environmental protozoa. The bacteria replicate in a specialized phagosome termed the Legionella-containing vacuole (LCV). Lpn exit of infected host cells may firstly involve release from the LCV and secondly release from the host cell. Exit is a principal step during infection allowing pathogen transmission and therefore is essential for continuation of disease or spread in the environment. But the mechanisms leading to release of the bacteria from the host are to a big extent not clear. Particularly bacterial phospholipases due to their membrane destructive capacity may support egress of Lpn. Lpn harbors a plenty of them comprising 15 phospholipases A (PLA), three phospholipases C (PLC), and a phospholipase D. The PLAs, especially the subgroups of GDSL and PlaB enzymes, and the PLCs due to their substrate specificity and high-level activity, abundance at later time points of growth, activation mode, secreted or bacterial surface-associated nature seem well suited to support bacterial egress. Indeed, some of those were already shown to have an impact in host exit. In the here anticipated project phase II, we will carry on our work on the basis laid by the results of project phase I which among others involve characterization of diverse phospholipase activation modes, phospholipase membrane destructive capacity, establishment of LCV isolation and lipidomics analysis, and identification of suitable bacterial surface proteins for egress reporter presentation. Accordingly, we will continue to 1) establish methods for efficient Lpn egress quantification, such as a beta-lactamase- or a sphingomyelin reporter-based assay for sensing cytosolic release of bacteria combined with fluorescence readout detection. 2) We will study the importance of Lpn phospholipases and respective hyperactive versions for Lpn exit by means of determining exit kinetics of defined Lpn mutants. 3) We will continue to investigate how Lpn phospholipases may support egress from the LCV and which lipids hydrolyzed are critical for the phenotypes observed. Further, we will apply artificial lipid bilayer assays to determine the phospholipase enzymatic potential for membrane permeabilization. 4) We will use an unbiased Lpn screen for essential exit genes involving the Tn-seq method. The mutants of interest will be further characterized respective grade and quality of exit defect, effect on LCV lipids, and for their behavior in different infection models. In summary, data obtained by the described experiments will help to characterize the egress process of Lpn in more detail.

嗜肺军团菌是一种重要的细胞内致病菌，可感染人体肺部和环境中的原虫。这种细菌在一种特殊的吞噬小体中复制，这种吞噬小体被称为含有军团菌的液泡(LCV)。受感染的宿主细胞可能首先从LCV中释放LPN，然后再从宿主细胞中释放。出口是在感染过程中允许病原体传播的主要步骤，因此对于疾病的延续或在环境中传播是必不可少的。但导致细菌从宿主释放的机制在很大程度上尚不清楚。尤其是细菌磷脂酶，由于其膜的破坏能力，可能支持LPN的出口。LPN含有大量的磷脂酶，包括15个磷脂酶A(PLA)、3个磷脂酶C(PLC)和1个磷脂酶D。PLAs，特别是GDSL和PLAB酶的亚群，以及PLCs由于其底物特异性和高水平的活性，在生长后期丰富，激活方式，分泌性或细菌表面相关的性质，似乎非常适合支持细菌出口。事实上，其中一些已经被证明对东道国退出产生了影响。在这里预期的项目第二阶段，我们将在项目第一阶段结果的基础上继续我们的工作，其中包括表征不同的磷脂酶激活模式，磷脂酶膜的破坏能力，建立LCV分离和脂质组学分析，以及鉴定适合出口记者展示的细菌表面蛋白。因此，我们将继续1)建立有效的LPN出口定量方法，例如基于β-内酰胺酶或鞘磷脂报告基因的检测方法，用于检测细菌的胞浆释放，并结合荧光读数检测。2)我们将通过确定已定义的LPN突变体的退出动力学来研究LPN磷脂酶及其各自的高活性版本对LPN退出的重要性。3)我们将继续研究LPN磷脂酶如何支持LCV的出口，以及哪些脂类被水解对所观察到的表型至关重要。此外，我们将应用人工脂双层分析来确定磷脂酶对膜通透性的潜力。4)我们将使用无偏LPN筛选必要的退出基因，包括TN-SEQ方法。感兴趣的突变体将进一步表征各自的出口缺陷的级别和质量，对LCV血脂的影响，以及它们在不同感染模式中的行为。总之，通过所描述的实验获得的数据将有助于更详细地描述LPN的出射过程。