SummaryThe intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella , the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two-hybrid screen. It was determined that the IcmS-IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS-IcmW complex being involved in the recognition and Dot/ Icm-dependent translocation of substrate proteins during Legionella infection of host cells.

No abstract

Proteins of the Smr family are the smallest multidrug transporters, about 110 amino acids long, that extrude various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. One of these proteins, EmrE, is an Escherichia coli protein, which has been cloned based on its ability to confer resistance to ethidium and methyl viologen and which has been extensively characterized. More than 60 genes coding for Smr proteins have been identified in several bacteria based on amino acid sequence similarity to the emrE gene. In this work we have analyzed the sequence similarity among these homologues and identified some distinct signature sequence elements and several fully conserved residues. Five of these homologues, from human pathogens Mycobacterium tuberculosis, Bordetella pertussis, and Pseudomonas aeruginosa and from Escherichia coli, were cloned into an E. coli expression system. The proteins were further characterized and show varying degrees of methyl viologen uptake into proteoliposomes and

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.

EmrE is a multidrug transporter from Escherichia coli that functions as a homooligomer and is unique in its small size. In each monomer there are four tightly packed transmembrane segments and one membrane-embedded charged residue. This residue provides the basis for the coupling mechanism as part of a binding site "time shared" by substrates and protons.