Function of Legionella pneumophila phospholipases in vacuole and host egress

• Applicant: Professorin Dr. Antje Flieger
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 446411117

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.

• DFG Programme: Priority Programmes
• Subproject of: SPP 2225: Exit strategies of intracellular pathogens