Legionella pneumophila, the etiological agent for the potentially fatal Legionnaires'disease, is a facultative intracellular pathogen. Intracellular replication of L. pneumophila in phagocyte is entirely dependent upon the Dot/Icm type IV secretion system, which translocates hundreds of virulence factors (effectors) into host cells. These effectors directly modulate host cellular functions to create a permissive niche that supports bacterial growth within hostile phagocytic cells. Mutants lacking individual effector genes rarely display significant defect in intracellular growth of the bacterium, making it difficut to obtain a complete understanding of their role in infection. Several features associated with the Dot/Icm substrate SidJ make it a very useful lead to dissect specific bacterial activity important for L. pneumophila intracellular replication. First, a sidJ deletion mutant was defective in intracellular growth in both mouse bone marrow-derived macrophages and the amoebae host Dictyostelium discoideum. Further, acquisition of endoplasmic reticulum proteins such as calnexin by vacuoles harboring the sidJ deletion mutant was delayed. Second, the toxicity of SidJ to yeast under overexpression condition was due to the titration of calmodulin. In an independent line of study aiming at analyzing the impact of Dot/Icm substrates on eukaryotic cells, we found that SdeA strongly inhibits yeast growth and the secretory pathway in mammalian cell. Further, we found that several yeast proteins involved in membrane transport between the endoplasmic reticulum and the cis-Golgi apparatus can suppress the toxicity of SdeA. More importantly, in a screening to search for L. pneumophila protein that may regulate SdeA activity, we identified SidJ as a strong suppressor of the toxicity of SdeA to yeast and of its inhibitory effect on mammalian secretion. Thus, we hypothesized that SdeA modulates membrane transport between the endoplasmic reticulum (ER) and the Golgi apparatus and that SidJ regulates such modulation. We will test this hypothesis by pursuing three specific aims:
Aim 1 : Determine the cellular target and the biochemical function of SdeA. The working hypothesis is that SdeA targets one or more host proteins involved in membrane transport between the endoplasmic reticulum and the cis-Golgi compartment. We will determine the host protein targeted by SdeA and whether and how SdeA biochemically modifies the host protein. We will also determine how such modification alters the function of the host protein.
Aim 2 : Analyze the regulation of SdeA activity by SidJ. The working hypothesis is that SidJ functions to safeguard the effects of SdeA by temporally and/or spatially regulating its activity. We will determine the biochemical activity of SidJ and the role of such activity in the regulation of SdeA function.
Aim 3 : Assess the relationship between SdeA (and members of the SidE family) and other Dot/Icm substrates involved in ER-Golgi transport. The working hypothesis is that SdeA/SidJ function in concert with other Dot/Icm substrates involved in modulating host membrane trafficking to divert the transport of membrane materials to the bacterial phagosome. We will first obtain a set of Dot/Icm substrates involved in ER-Golgi transport by their ability to inhibit secretion by mammalian cells, followed by the construction of mutants lacking these genes for evaluation of their roles in intracellular bacterial replication. These studies will lay he foundation for the investigation in the modulation of host vesicle trafficking by bacterial virulene factors and will have translational implications in the context of treatment and prevention of Legionella infections.
Infections caused by Legionella pneumophila are a significant concern in public health. Equally important is that L. pneumophila has become one of the best pathogen models in study host cell biology under infection conditions;knowledge learned from this system can provide leads in our study of other diseases associated with abnormalities of cellular processes identified or better understood by studying this organism. Thus, our study can provide novel strategies for the prevention and/or treatment of infections caused by Legionella pneumophila and other important pathogens!
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