Function of Legionella pneumophila phospholipases in vacuole and host egress

嗜肺军团菌（Legionella pneumophila, Lpn）是一种重要的细胞内致病菌，感染人类肺部和环境原生动物。细菌复制在一个特殊的吞噬体称为含军团菌液泡（LCV）。感染宿主细胞的Lpn退出可能首先涉及LCV的释放，其次是宿主细胞的释放。退出是感染期间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至关重要。但导致细菌从宿主中释放的机制在很大程度上尚不清楚。特别是细菌磷脂酶，由于其膜破坏能力，可能支持Lpn的排出。Lpn含有大量的磷脂酶，包括15种磷脂酶a （PLA）、3种磷脂酶C （PLC）和1种磷脂酶d。这些磷脂酶，尤其是GDSL和PlaB酶亚群，由于它们的底物特异性和高活性，在生长后期的时间点上的丰度，激活模式，分泌或细菌表面相关的性质，似乎非常适合支持细菌的出口。事实上，其中一些已经被证明对主机退出有影响。在这里预期的项目二期中，我们将在项目一期成果的基础上继续我们的工作，其中包括不同磷脂酶激活模式的表征，磷脂酶膜破坏能力，LCV分离和脂质组学分析的建立，以及适合出口报告的细菌表面蛋白的鉴定。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蛋白的检测，用于检测细菌的胞质释放，并结合荧光读数检测。2)我们将通过确定定义的Lpn突变体的退出动力学来研究Lpn磷脂酶和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的输出，以及哪些脂质水解对观察到的表型至关重要。此外，我们将应用人工脂质双分子层测定法来确定磷脂酶对膜渗透的酶促电位。4)我们将使用无偏Lpn筛选涉及n-seq方法的必要退出基因。研究人员将进一步描述这些突变体各自的出口缺陷的等级和质量、对LCV脂质的影响以及它们在不同感染模型中的行为。总之，所描述的实验获得的数据将有助于更详细地表征Lpn的输出过程。

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.