Exploitation of host glycolysis by Legionella

University Of Louisville, Louisville KY

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Abstract

? DESCRIPTION (provided by applicant): Upon transmission to humans, Legionella pneumophila (Lp) proliferates within alveolar macrophages, causing pneumonia with high morbidity and mortality. Intracellular proliferation of Lp occurs within the endoplasmic reticulum-derived Legionella-containing vacuole (LCV), which evades lysosomal fusion. Biogenesis of the LCV is governed by the Dot/Icm type IV secretion system, which injects into the host cell ~300 protein effectors that exploit a myriad of cellular processes to enable intra- vacuolar proliferatin of Lp. The generation time of intra-vacuolar Lp within human macrophages is ~40 min, which is 3-4 times faster than growth in rich media. Amino acids are the major sources of carbon and energy for Lp, which metabolize them through the TCA cycle as the major energy-yielding metabolic pathway in Lp, which does not have a functional glycolytic pathway. However, our recent studies indicate that the host basal cellular levels of amino acids are below the threshold needed as a major source of carbon and energy to feed the TCA cycle of intra-vacuolar Lp. To achieve that threshold, Lp promotes host proteasomal degradation, which is totally bypassed upon supplementation of amino acids or pyruvate. This indicates the import of pyruvate by the LCV and its utilization by intra-vacuolar Lp as a source of carbon and energy. Our preliminary data show a Dot/Icm-dependent up-regulation of the host glycolytic enzymes and production of pyruvate in Lp-infected human monocytes-derived macrophages (hMDMs). Importantly, glucose deprivation or inhibition of glycolysis in hMDMs suppresses intra-vacuolar proliferation of Lp, but the suppression is totally relieved upon supplementation of pyruvate. This indicates that the LCV imports pyruvate to be utilized by intra-vacuolar Lp as a source of carbon and energy. Our findings indicate a multi-prong bacterial strategy to retrieve diverse host resources to feed the TCA cycle of Lp. The import of pyruvate across eukaryotic membranes is mediated by the plasma membrane-localized SLC16A1 and SLC16A7 transporters and by the mitochondrial membrane-localized MPC1 and MPC2 oligomeric complex. Our preliminary data derived from microarrays of Lp-infected hMDMs show that Lp triggers Dot/Icm-dependent up-regulation of the host MPC1 and MPC2, but not SLC16A1 or SLC16A7. Our profiling of the proteome of the LCV membrane shows that the MPC1 and MPC2 eukaryotic transporter is acquired by the LCV in a Dot/Icm-dependent manner, but the SLC16A1 and SLC16A7 are not detectable in the LCV proteome. Therefore, our hypothesis is: the host MPC pyruvate transporter is acquired by the LCV membrane through interaction with a Dot/Icm-translocated effector to import pyruvate into the LCV to be utilized by intra-vacuolar Lp as a source of carbon and energy. To test our hypothesis, our specific aims are to determine and characterize the following: I. Acquisition of host MPC transporter by the LCV and its import of pyruvate as a source of carbon and energy for intra-vacuolar Lp; II. The Lp effectors that interact with the host MPC transporter in the LCV membrane. The significance of our studies stem from deciphering exploitation of host glycolysis by intra- vacuolar Lp; and determination of the mechanisms of import of host pyruvate from the cytosol of primary hMDMs into a pathogen-containing vacuole and its utilization by the pathogen as a source of carbon and energy. The innovations of our studies stem from: Novelty of the hypotheses; cutting-edge technologies and experimental design; Dot/Icm-dependent up-regulation of host glycolysis, production of pyruvate; and up- regulation of the host MPC1 and MPC2 transporter; recruitment of the host MPC1 and MPC2 transporter into the pathogen-containing vacuole; import of pyruvate generated from host glycolysis by the pathogen- containing vacuolar membrane; and identification of novel bacterial effectors that interact with the host MPC1 and MPC2 transporter in the vacuolar membrane of an intra-vacuolar pathogen.