Saudi Cultural Missions Theses & Dissertations
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Item Restricted Legionella pneumophila Infection and The Host Unfolded Protein Response(Monash University, 2024) Alshareef, Manal Hashim; Hartland, Elizabeth; McCaffrey, KathleenLegionella pneumophila is a Gram-negative bacterium that survives in the environment by replicating within free-living amoebae. When transmitted to humans through contaminated aerosols, the pathogen infects phagocytic immune cells within the lung, such as macrophages and monocytes, to cause disease. This respiratory disease features either severe pneumonia, known as Legionnaires’ disease, or a milder infection called Pontiac fever. To survive and replicate within a eukaryotic cell, Legionella species use a type-IVB secretion system, termed Dot/Icm, to secrete >330 “effector” proteins into the host cell. Dot/Icm effectors manipulate various host processes to evade elimination by phago-lysosomal degradation and establish an intracellular replication vacuole, termed the Legionella-containing vacuole (LCV). A key feature of the LCV is its similarity to rough endoplasmic reticulum (ER) membranes raising the possibility that Legionella induces ER stress and the unfolded protein response (UPR). The UPR is a homeostatic response to ER stress that can play an important role in infection and immunity. L. pneumophila Dot/Icm effectors, including Lgt1-3, SidI, and SidL, have been previously shown to inhibit UPR signalling by blocking host cell protein synthesis. However, whether the UPR restricts L. pneumophila replication or modulates the host immune response to Legionella infection remains unknown. Here we demonstrated that L. pneumophila infection of a macrophage THP-1 cell line induces host ER stress and activates canonical UPR signalling via IRE1, PERK, and ATF6. This activation is a Dot/Icm-dependent. Using pharmacological inhibitors of UPR signalling, we also demonstrated that IRE1 RNase activity supports L. pneumophila intracellular replication in THP-1 macrophages. In contrast, pre-treatment of THP-1 macrophages with pharmacological inducers of ER stress, tunicamycin and thapsigargin prior to infection reduced L. pneumophila intracellular replication. Drug pre-treatment did not inhibit L. pneumophila growth in vitro, phagocytic uptake of the bacterium or Dot/Icm effector translocation. Although tunicamycin enhanced cell death resulting in reduced bacterial load, thapsigargin pre-treatment instead protected macrophages from L. pneumophila-induced cytotoxicity. Thapsigargin induced restriction of L. pneumophila intracellular replication relied on IRE1-kinase activity and STAT1 activation, and hence was linked to UPR-mediated immunity during ER stress. How this restriction is orchestrated needs to be further investigated. Finally, we successfully constructed a L. pneumophila mutant of strain 130b lacking Lgt1, Lgt3, SidI, and SidL, which we termed delta4. The delta4 mutant exhibited normal in vitro growth and was not different from the wild-type parent strain in terms of intracellular replication withinTHP-1 macrophages and loss of Lgt1, Lgt3, SidI, and SidL partially restored host protein synthesis and IRE1-dependent XBP1 mRNA splicing, similar to previous studies with L. pneumophila strain Philadelphia. The delta4 mutant inhibited UPR signalling in THP-1 cells early during the infection (~6 h) but not later in the replicative phase of the infection. Interestingly, wild-type L. pneumophila 130b inhibited STAT1 signalling compared to the delta4 mutant, which induced ER stress in THP-1 macrophages suggesting that Dot/Icm effectors play a role in modulating the host immune response induced by ER stress during L. pneumophila infection.17 0