A Legionella pneumophila Effector Protein Encoded in a Region of Genomic Plasticity Binds to Dot/Icm-Modified Vacuoles

Shira Ninio; Jean Celli; Craig R Roy

doi:10.1371/journal.ppat.1000278

Legionella pneumophila is an opportunistic pathogen that can cause a severe pneumonia called Legionnaires' disease. In the environment, L. pneumophila is found in fresh water reservoirs in a large spectrum of environmental conditions, where the bacteria are able to replicate within a variety of protozoan hosts. To survive within eukaryotic cells, L. pneumophila require a type IV secretion system, designated Dot/Icm, that delivers bacterial effector proteins into the host cell cytoplasm. In recent years, a number of Dot/Icm substrate proteins have been identified; however, the function of most of these proteins remains unknown, and it is unclear why the bacterium maintains such a large repertoire of effectors to promote its survival. Here we investigate a region of the L. pneumophila chromosome that displays a high degree of plasticity among four sequenced L. pneumophila strains. Analysis of GC content suggests that several genes encoded in this region were acquired through horizontal gene transfer. Protein translocation studies establish that this region of genomic plasticity encodes for multiple Dot/Icm effectors. Ectopic expression studies in mammalian cells indicate that one of these substrates, a protein called PieA, has unique effector activities. PieA is an effector that can alter lysosome morphology and associates specifically with vacuoles that support L. pneumophila replication. It was determined that the association of PieA with vacuoles containing L. pneumophila requires modifications to the vacuole mediated by other Dot/Icm effectors. Thus, the localization properties of PieA reveal that the Dot/Icm system has the ability to spatially and temporally control the association of an effector with vacuoles containing L. pneumophila through activities mediated by other effector proteins. The survival of intracellular pathogens often involves the modification of the host vacuole in which the pathogen resides. This can be achieved through the function of effector proteins that are delivered into the host cell cytoplasm using specialized transport machinery. In the case of Legionella pneumophila , the bacterium that causes a severe pneumonia known as Legionnaires' disease, a type IV secretion system, termed Dot/Icm, delivers a number of proteins into host cells, resulting in altered trafficking of the L. pneumophila –containing vacuole. The mechanisms by which effector proteins are spatially and temporally regulated in the host cell to facilitate the survival of the pathogen are not well understood. In this work, we report the identification of several L. pneumophila effectors encoded in a genomic region of high plasticity, among them the protein PieA. We demonstrate the Dot/Icm dependent recruitment of PieA to the L. pneumophila vacuole and show that the protein binds to the cytoplasmic face of the vacuole as a result of L. pneumophila –induced modifications to this vacuole. Our findings demonstrate that the association of an effector with host vacuoles can be spatially controlled through activities mediated by other effector proteins.

A Legionella pneumophila Effector Protein Encoded in a Region of Genomic Plasticity Binds to Dot/Icm-Modified Vacuoles

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