Examining the localization of transiently expressed and translocated Legionella pneumophila effector proteins in mammalian cells

• Samantha Sharp, Biological Sciences, University of Delaware

• Ramona Neunuebel, Biological Sciences, University of Delaware

Legionella pneumophila is an intracellular bacterial pathogen that infects alveolar macrophages and causes a severe form of pneumonia known as Legionnaires’ disease. The bacterium translocates 330+ effector proteins through a specialized secretion system to manipulate host cell pathways. Some of these effectors bind to phosphoinositides (PIPs), phospholipids that regulate membrane identity and intracellular trafficking. These PIP-binding effectors recruit host membranes and proteins to its Legionella-containing vacuole, enabling the vacuole to resemble host organelles and evade lysosomal degradation. Although hundreds of effector proteins have been identified, many of their functions and subcellular localizations remain poorly defined. Our primary goal is to examine the colocalization of uncharacterized PIP-binding effector proteins with specific host targets in mammalian cells under different conditions. 

We first sought to validate the effectiveness of our new polyclonal antibodies, which originated from animals inoculated with purified effector proteins. We used immunoblots to verify inducible expression of effector proteins fused with an HA-tag and compared this to blots probed with the polyclonal antibodies. Next, human cells were transfected with plasmids expressing fluorescently-tagged effector proteins or infected with wild-type, translocation-deficient, or HA-tagged effector-expressing Legionella strains. Immunostaining and fluorescence microscopy were then performed to visualize the subcellular colocalization of the PIP-binding effector proteins with their candidate host targets during transfection and infection.

We found that the polyclonal antibodies detected the effector proteins in immunoblots and were, in some cases, suitable for immunostaining of infected cells. We observed colocalization between PIP-binding effectors and their suspected localization targets, and overlap between signals for the HA-tag and the polyclonal effector antibodies. 

These findings demonstrate that the polyclonal antibodies are suitable for observation of effector localization during transfection and infection, which will aid in defining the targets and function of these poorly understood effector proteins and provide insight into the pathogenicity of Legionella.