Translating inflammation: characterization of host protein synthesis during bacterial infections

Translating inflammation: characterization of host protein synthesis during bacterial infections/nby/nKevin Christopher Barry/nDoctor of Philosophy in Molecular and Cell Biology/nUniversity of California, Berkeley/nProfessor Russell E. Vance, Chair/nThe innate immune system is the first line of defense against pathogens. Innate immune receptors, termed pattern recognition receptors, are germline-encoded receptors that recognize conserved microbial products and activate an immune response. Examples of these microbial products, termed pathogen-associated molecular patterns, are components of the bacterial outer membrane, such as lipopolysaccharide or bacterial lipoproteins, and microbe-derived nucleic acids. Importantly these molecular patterns are not just found on pathogens, but are also encoded by harmless commensal microbes as well. It has become clear in recent years that the innate immune system distinguishes pathogens from harmless commensals and preferentially responds to pathogens. It has been established that one mechanism by which the innate immune system makes this distinction is through the recognition of activities that are associated with the pathogenic lifestyle, termed patterns of pathogenesis, such as access to the host cytosol and microbial growth. Recently, translation inhibition induced by pathogenic microbes has been shown to be important for the induction of immune responses, and thus has been termed a novel pathogen-associated activity./nLegionella pneumophila is a gram-negative intracellular bacterial pathogen that is the causative agent of a severe pneumonia called Legionnaires’ Disease. After inhalation of aerosolized bacteria, L. pneumophila can infect and replicate within lung alveolar macrophages. Intracellular replication of L. pneumophila in macrophages in vitro, and virulence of L. pneumophila in animal models, requires a Type IV secretion system (T4SS) called the Dot/Icm system, which secretes bacterial effector proteins into the host cytosol. These effectors, greater than 270 of which have been identified, are believed to be critical for establishment of the Legionella-containing vacuole, the specialized membrane-bound intracellular compartment in which L. pneumophila replicates. In addition to its essential role in facilitating intracellular bacterial replication, the L. pneumophila T4SS is also associated with a strong block in host protein synthesis and the induction of several potent innate immune responses./nThe over-arching goal of this thesis is to expand our knowledge of the mechanisms by which the innate immune system distinguishes pathogenic microbes from non-pathogenic microbes. In the first chapter of this thesis I will review the current state of the field. In the second chapter of this thesis I will describe studies using L. pneumophila infection in vivo where I found an important role for the often-overlooked cytokine, interleukin-1α (IL-1α), in initiating the immune response to virulent L. pneumophila. I was able to demonstrate, consistent with previous studies, that signaling through the interleukin-1 receptor (IL-1R) is important for the recruitment of protective neutrophils to the lungs of mice, but unlike previous studies, we could show that the early recruitment of these cells required IL-1α. I was further able to characterize the molecular mechanism by which the innate immune system is able to produce IL-1α specifically in response to virulent infection. I found that host protein synthesis is inhibited by T4SS+, but not T4SS–, L. pneumophila. I was further able to show that translation inhibition in concert with signaling via the innate immune receptors the toll-like receptors (TLRs) induced sustained and massive induction of Il1a transcript. I proposed that this massive induction of Il1a transcript overcame the L. pneumophila induced block in host protein synthesis and permitted the enhanced production and release of IL-1α. Thus, these studies demonstrated that IL-1α, a cytokine I showed to be important for protecting the host from L pneumophila infection in vivo, was preferentially made in response to T4SS+ L. pneumophila. Moreover, I linked the production of IL-1α to the sensing of the pathogen-induced block in host protein synthesis. These studies also identified five known and two novel bacterial effectors that block host protein synthesis, but deletion of all seven of these effectors did not affect the L. pneumophila induced block in host protein synthesis. I hypothesized that other mechanisms, possibly host stress induced by intracellular bacterial infection, could induce this block in translation. Thus, taken together, the experiments described in the second chapter of this thesis identify a novel inflammatory response to L. pneumophila in vivo and further support a model in which pathogen-induced translation inhibition can allow the immune system to detect a pathogen and respond appropriately./nIn the third chapter of this thesis I set out to further characterize the molecular mechanism of IL-1α production and translation inhibition induced by T4SS+ L. pneumophila. As deletion of the seven L. pneumophila effectors that block host protein synthesis did not relieve the block in host protein synthesis induced by L. pneumophila, I set out to determine if the residual block in host protein synthesis by the Δ7 L. pneumophila mutant was at the level of translation initiation or elongation. Using a deep sequencing technique called ribosome profiling in concert with RNAseq of total mRNA, I was able to look at translation in L. pneumophila infected macrophages globally and with nucleotide resolution. I found through these analyses that T4SS+ L. pneumophila blocks translation elongation, but the residual translation inhibition induced by Δ7 L. pneumophila was at the level of translation initiation. The vast majority of translational control by the host is at the level of translation initiation. Thus, the Δ7 L. pneumophila induced block in translation initiation suggests that a host stress response could be blocking translation in response to the stresses of being infected by an intracellular pathogen. In the third chapter of this thesis I assay a number of host stress response pathways after L. pneumophila infection and see no role for these pathways in Δ7 L. pneumophila induced translation inhibition. I proposed that these data suggest that an unknown stress response pathway may be activated or, alternatively, a novel bacterial effector could be blocking translation initiation./nThe studies described in the third chapter of this thesis also undertook analyses of ribosome profiling and RNAseq data to further test the model that inflammatory cytokines are made in response to pathogens by the massive induction of transcripts in response to the pathogen-associated activity of blocking host protein synthesis along with TLR signaling. The data presented in the third chapter support a model that the induction of cytokine transcripts via sensing of the pathogen-associated activity of translation inhibition and TLR activation overcomes the block in host protein synthesis and allows the infected cell to preferentially respond to pathogens with the production of inflammatory cytokines. I further describe experiments that suggest diverse intracellular bacterial pathogens such as Listeria monocytogenes also induce a block in host protein synthesis and that this activity may be a broadly applicable pathogen-associated activity. Lastly, the studies presented in the third chapter of this thesis provide evidence that, at least in response to virulent L. pneumophila, the majority of control of gene expression in response to pathogenic infection is controlled at the level of mRNA induction./nThe studies presented in this thesis lend credence to the proposal that translation inhibition is a pathogen-associated activity encoded by diverse intracellular bacterial pathogens. They also support a model by which translation inhibition is sensed by host innate immune cells to induce massive mRNA induction of inflammatory cytokines allowing for a specific inflammatory response to pathogens. Lastly, these studies link translation inhibition to an important role in protecting the host from pathogenic infection in vivo.

Advisor: Vance, Russell E