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Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens
Published on: October 25, 2019
Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking
Nadim Shohdy1, Jem A Efe, Scott D Emr
1Department of Microbiology, Columbia University Medical Center, 701 West 168th Street, New York, NY 10032, USA.
Abstract:
Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.
Insights
Legionella pneumophila uses effector proteins to disrupt host cell organelle trafficking. This study identified novel bacterial proteins that inhibit vacuole protein sorting (VPS) and lysosomal pathways, revealing new virulence strategies.
Area of Science:
- Microbiology
- Cell Biology
- Genetics
Background:
- Legionella pneumophila is an intracellular pathogen that manipulates host cell processes.
- The Icm/Dot type IVB secretion system is crucial for translocating bacterial effectors into host cells.
- Understanding how L. pneumophila effectors disrupt host trafficking is key to deciphering its virulence.
Purpose of the Study:
- To identify novel Legionella pneumophila effector proteins involved in modulating host cell membrane trafficking.
- To investigate the mechanisms by which these effectors perturb vacuole protein sorting (VPS) and organelle transport.
Main Methods:
- Utilized a high-throughput yeast-based screening assay to identify L. pneumophila genes causing VPS defects.
- Characterized the function of identified effector proteins (VipA, VipD, VipF) in yeast and mammalian cells.
- Investigated the impact of effectors on specific trafficking pathways, including lysosomal protein transport and endosome-to-Golgi transport.
Main Results:
- Identified four L. pneumophila DNA fragments that disrupt vacuolar protein sorting in yeast.
- Three fragments encode novel effector proteins (VipA, VipD, VipF) translocated into macrophages.
- VipA, VipD, and VipF inhibit lysosomal protein trafficking through distinct mechanisms, affecting carboxypeptidase Y trafficking, multivesicular body formation, and ER-to-Golgi transport.
Conclusions:
- Yeast serves as an effective model for discovering bacterial virulence factors and studying their functions.
- L. pneumophila employs diverse effector strategies to subvert host organelle trafficking pathways.
- The identified Vip proteins represent novel tools for dissecting host-pathogen interactions and cellular trafficking mechanisms.
