Characterizing PIP-binding Legionella Pneumophila effector proteins using Halo-tag and GFP constructs

• Adam Kerzner, Biological Sciences, University of Delaware

• Ramona Neunuebel, Biological Sciences, University of Delaware

Legionella pneumophila (hereafter Legionella) is an intracellular Gram-negative bacterial pathogen that infects alveolar macrophages and causes an acute pneumonia known as Legionnaires’ disease. Legionella avoids degradation by macrophages through the establishment of the Legionella-Containing Vacuole (LCV) through employment of its Type IV secretion system to translocate effector proteins. Many of these effector proteins target and bind to phosphoinositides (PIPs), which play a crucial role in vesicular trafficking as different membranes in the cell are enriched in different species of PIPs. Here we investigate these novel PIP-binders through generating Halo-tagged and GFP-tagged effector protein constructs to study effector proteins Lpg1884, Lpg2511, and Lpg1958. GFP (Green Fluorescent Protein) constructs will be expressed in mammalian cells to determine the effector protein’s cellular distribution. Halo-tag fusion constructs will be expressed in and subsequently used to infect host cells to determine where effector proteins are localized after secretion into the host cell. To create these Halo-tag and GFP-tag fusion constructs, I used Gateway Cloning to clone the DNA encoding these proteins into a plasmid that comprised the gene for the Halo tag or the GFP tag. I then transformed these plasmids into competent 2T1 cells and transformed the Halo tag constructs into Legionella. Using colony PCR, gel electrophoresis, and western blotting, I verified the constructs and confirmed the correct protein expression. The next step is to infect mammalian cells with Legionella over-expressing the Halo tag constructs and transfect mammalian cells with GFP tagged constructs. This will allow us to know when and where these PIP-binding proteins are secreted, and if they directly bind PIPs or interact with other compartments in the cell. Understanding the function of these PIP-binding proteins will provide a greater comprehension of how Legionella is able to survive in human cells by manipulating the host cell’s vesicular trafficking pathways and avoiding degradation.