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Open Access Publications from the University of California

Scholarly Works (14 results)

  • Article
  • Peer Reviewed
Proceedings of the Annual Meeting of the Cognitive Science Society, Volume 36 (2014)
    Cover page: Potential Mediators of Perceptual-motor Performance Degradation Resulting from Increased Cognitive Workload
    • Thesis
    • Peer Reviewed
    UC Berkeley Electronic Theses and Dissertations (2015)

    Translating inflammation: characterization of host protein synthesis during bacterial infections

    by

    Kevin Christopher Barry

    Doctor of Philosophy in Molecular and Cell Biology

    University of California, Berkeley

    Professor Russell E. Vance, Chair

    The 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.

    Legionella 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.

    The 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.

    In 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.

    The 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.

    The 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.

      Cover page: Translating inflammation: characterization of host protein synthesis during bacterial infections
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2017)
      The inducible innate immune response to infection requires a concerted process of gene expression that is regulated at multiple levels. Most global analyses of the innate immune response have focused on transcription induced by defined immunostimulatory ligands, such as lipopolysaccharide. However, the response to pathogens involves additional complexity, as pathogens interfere with virtually every step of gene expression. How cells respond to pathogen-mediated disruption of gene expression to nevertheless initiate protective responses remains unclear. We previously discovered that a pathogen-mediated blockade of host protein synthesis provokes the production of specific pro-inflammatory cytokines. It remains unclear how these cytokines are produced despite the global pathogen-induced block of translation. We addressed this question by using parallel RNAseq and ribosome profiling to characterize the response of macrophages to infection with the intracellular bacterial pathogen Legionella pneumophila. Our results reveal that mRNA superinduction is required for the inducible immune response to a bacterial pathogen.
        Cover page: Global analysis of gene expression reveals mRNA superinduction is required for the inducible immune response to a bacterial pathogen
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2014)
        Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (HSL-C12) as a quorum-sensing molecule to regulate bacterial gene expression. Because HSL-C12 is membrane permeant, multiple cell types in P. aeruginosa-infected airways may be exposed to HSL-C12, especially adjacent to biofilms where local (HSL-C12) may be high. Previous reports showed that HSL-C12 causes both pro- and anti-inflammatory effects. To characterize HSL-C12's pro- and anti-inflammatory effects in host cells, we measured protein synthesis, NF-κB activation, and KC (mouse IL-8) and IL-6 mRNA and protein secretion in wild-type mouse embryonic fibroblasts (MEF). To test the role of the endoplasmic reticulum stress inducer, PERK we compared these responses in PERK(-/-) and PERK-corrected PERK(-/-) MEF. During 4-h treatments of wild-type MEF, HSL-C12 potentially activated NF-κB p65 by preventing the resynthesis of IκB and increased transcription of KC and IL-6 genes (quantitative PCR). HSL-C12 also inhibited secretion of KC and/or IL-6 into the media (ELISA) both in control conditions and also during stimulation by TNF-α. HSL-C12 also activated PERK (as shown by increased phosphorylation of eI-F2α) and inhibited protein synthesis (as measured by incorporation of [(35)S]methionine by MEF). Comparisons of PERK(-/-) and PERK-corrected MEF showed that HSL-C12's effects were explained in part by activation of PERK→phosphorylation of eI-F2α→inhibition of protein synthesis→reduced IκBα production→activation of NF-κB→increased transcription of the KC gene but reduced translation and secretion of KC. HSL-C12 may be an important modulator of early (up to 4 h) inflammatory signaling in P. aeruginosa infections.
          Cover page: Pseudomonas aeruginosa Quorum-Sensing Molecule Homoserine Lactone Modulates Inflammatory Signaling through PERK and eI-F2α
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2020)
          Natural killer (NK) cells belong to the innate immune system and contribute to protecting the host through killing of infected, foreign, stressed or transformed cells. Additionally, via cellular cross-talk, NK cells orchestrate antitumor immune responses. Hence, significant efforts have been undertaken to exploit the therapeutic properties of NK cells in cancer. Current strategies in preclinical and clinical development include adoptive transfer therapies, direct stimulation, recruitment of NK cells into the tumor microenvironment (TME), blockade of inhibitory receptors that limit NK cell functions, and therapeutic modulation of the TME to enhance antitumor NK cell function. In this Review, we introduce the NK cell-cancer cycle to highlight recent advances in NK cell biology and to discuss the progress and problems of NK cell-based cancer immunotherapies.
            Cover page: The NK cell–cancer cycle: advances and new challenges in NK cell–based immunotherapies
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2016)
            The rising success of cancer immunotherapy has produced immense interest in defining the clinical contexts that may benefit from this therapeutic approach. To this end, there is a need to ascertain how the therapeutic modulation of intrinsic cancer cell programs influences the anticancer immune response. For example, the role of autophagy as a tumor cell survival and metabolic fitness pathway is being therapeutically targeted in ongoing clinical trials that combine cancer therapies with antimalarial drugs for the treatment of a broad spectrum of cancers, many of which will likely benefit from immunotherapy. However, our current understanding of the interplay between autophagy and the immune response remains incomplete. Here, we have evaluated how autophagy inhibition impacts the antitumor immune response in immune-competent mouse models of melanoma and mammary cancer. We observed equivalent levels of T cell infiltration and function within autophagy-competent and -deficient tumors, even upon treatment with the anthracycline chemotherapeutic doxorubicin. Similarly, we found equivalent T cell responses upon systemic treatment of tumor-bearing mice with antimalarial drugs. Our findings demonstrate that antitumor adaptive immunity is not adversely impaired by autophagy inhibition in these models, allowing for the future possibility of combining autophagy inhibitors with immunotherapy in certain clinical contexts.
              Cover page: Antitumor adaptive immunity remains intact following inhibition of autophagy and antimalarial treatment
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2011)
              The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires' Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs), leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR), requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also be shaped by pathogen-encoded activities, such as inhibition of host protein synthesis.
                Cover page: Secreted Bacterial Effectors That Inhibit Host Protein Synthesis Are Critical for Induction of the Innate Immune Response to Virulent Legionella pneumophila
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2017)
                All pathogens must acquire nutrients from their hosts. The intracellular bacterial pathogen Legionella pneumophila, the etiological agent of Legionnaires' disease, requires host amino acids for growth within cells. The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved master regulator of host amino acid metabolism. Here, we identify two families of translocated L. pneumophila effector proteins that exhibit opposing effects on mTORC1 activity. The Legionella glucosyltransferase (Lgt) effector family activates mTORC1, through inhibition of host translation, whereas the SidE/SdeABC (SidE) effector family acts as mTORC1 inhibitors. We demonstrate that a common activity of both effector families is to inhibit host translation. We propose that the Lgt and SidE families of effectors work in concert to liberate host amino acids for consumption by L. pneumophila.
                  Cover page: Positive and Negative Regulation of the Master Metabolic Regulator mTORC1 by Two Families of Legionella pneumophila Effectors
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2022)
                  The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte-macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.
                    Cover page: Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2022)
                    The Arctic is warming faster than anywhere else on Earth, prompting glacial melt, permafrost thaw, and sea ice decline. These severe consequences induce feedbacks that contribute to amplified warming, affecting weather and climate globally. Aerosols and clouds play a critical role in regulating radiation reaching the Arctic surface. However, the magnitude of their effects is not adequately quantified, especially in the central Arctic where they impact the energy balance over the sea ice. Specifically, aerosols called ice nucleating particles (INPs) remain understudied yet are necessary for cloud ice production and subsequent changes in cloud lifetime, radiative effects, and precipitation. Here, we report observations of INPs in the central Arctic over a full year, spanning the entire sea ice growth and decline cycle. Further, these observations are size-resolved, affording valuable information on INP sources. Our results reveal a strong seasonality of INPs, with lower concentrations in the winter and spring controlled by transport from lower latitudes, to enhanced concentrations of INPs during the summer melt, likely from marine biological production in local open waters. This comprehensive characterization of INPs will ultimately help inform cloud parameterizations in models of all scales.
                      Cover page: Annual cycle observations of aerosols capable of ice formation in central Arctic clouds
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