Characterizing the structure and function of phosphatidylinositol‐3‐phosphate‐binding L. pneumophilaeffectors

Abstract

Phosphoinositides (PIPs) are distinctly‐phosphorylated lipids that govern the fate of endocytosed cargo in eukaryotic vesicular trafficking. A growing number of pathogens manipulate these lipids to infect and survive within human cells. The intracellular bacterial pathogen L. pneumophila translocates a large number of effectors, some of which target PIPs to interfere with their metabolism or to anchor on to specific host membranes. This allows the phagocytosed bacterium to evade degradation and to proliferate inside host macrophages. Therefore, understanding how and why L. pneumophila effectors bind PIPs is crucial for uncovering their roles during infection. Most of the PIP‐binding effectors do not share homology with conserved eukaryotic PIP‐binding domains, making them difficult to identify and characterize. This work aims to identify the structures of the PIP binding regions for two effectors, Lpg2409 and Lpg0160, as well as to uncover the role of Lpg2409 during infection. We demonstrate that both effectors can be purified on a suitable scale for crystallography, and show that Lpg0160 has a modest binding affinity for a fluorescently‐labeled PI3P analog in vitro. Additionally, immunoprecipitation experiments with overexpressed mCherry‐Lpg2409 in HEK293T cells revealed binding to scaffolding proteins involved in E3 ubiquitin ligase complexes, as well as mitochondrial proteins, suggesting a role in regulating the turnover of mitochondrial proteins. These findings suggest that Lpg2409 may have a previously unidentified role during L. pneumophilainfection.