PlaD is a type IVB secreted effector phospholipase of Legionella pneumophila activated by host regulatory proteins

Introduction

Legionella pneumophila, the causative agent of a life-threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella-containing vacuole (LCV). During infection, L. pneumophila secretes proteins, among others phospholipases, into the lumen of the LCV and the host cell cytoplasm via its type II (Lsp) and type IVB (Dot/Icm) secretion systems. At least 15 phospholipases A, which divide into the patatin-like proteins, the PlaB-like proteins, and the GDSL hydrolases PlaA, PlaC and PlaD, are encoded in the genome.

Goals

We here characterize the phospholipase PlaD which shows various differences to the other GDSL hydrolases PlaA and PlaC. We aim to analyze the, activity and activation mechanism, secretion path, and importance and localization in infection of PlaD.

Materials and methods

The mode of secretion of PlaD was investigated by means of Western blotting and protein translocation assays. Additionally, we determined its binding to various lipids and its interactions with eukaryotic proteins by means of lipid-protein-overlay assays, proximity ligation, and pull-down assays. Further, we analyzed the localization of PlaD during infection via immunofluorescence microscopy.

Results

We found that, during infection, PlaD is Dot/Icm-dependently injected into the host cell cytoplasm where it localizes to distinct organelles. Moreover, PlaD binds to a subset of phosphoinositide species (PIPs) and interacts with a class of regulatory proteins of the host cell which leads to activation of phospholipase activity. Additionally, our data revealed that the C-terminal half of PlaD is essential for its secretion and phosphoinositide binding but dispensable for interaction with the regulatory proteins.

Summary

Based on its Dot/Icm dependent injection into the host cell cytoplasm, we classify PlaD as a novel type IVB secreted effector protein of L. pneumophila. Both its interaction with PIPs and eukaryotic proteins suggest a specific function in host cell infection.