Awardee Abstracts
Abstract:
Intracellular bacteria and protists are auxotrophic for many metabolites and must rely on the host cell to supply these nutrients. The mechanisms of how pathogens manipulate host metabolism to their benefit are not understood. These questions are difficult to address for intracellular pathogens because one cannot easily distinguish the origin of the metabolite as host or pathogen derived. Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90%. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells.
Toxoplasma gondii manipulates the host cell by a pre-invasion process called “kiss and spit”, where the contents of the parasite rhoptry organelles are secreted into the host cytoplasm before invasion occurs. Here, we demonstrated that rhoptry contents from kiss and spit altered metabolite abundance in nucleotide synthesis, the pentose phosphate pathway, glycolysis, and amino acid synthesis. An increase in 2,3-bisphosphoglycerate (2,3-BPG) abundance led us to investigate the activation of host cytosolic nucleosidase II (cN-II) to provide purines for the parasite.
Additionally, Optical metabolic Imaging (OMI) was used to analyze the fluctuations in redox biology in alive infected cells. I investigate how T. gondii manipulates the host cell metabolic environment by monitoring metabolic response over time using non-invasive autofluorescence lifetime imaging of single cells, seahorse metabolic flux analysis, reactive oxygen species (ROS) production, and metabolomics. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H with infection. These findings are consistent with changes in mitochondrial and glycolytic function, decrease of intracellular glucose, fluctuations in lactate and ROS production in infected cells over time. I also examined changes associated with the pre-invasion “kiss and spit” process using autofluorescence lifetime imaging, which similarly showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours. Glucose metabolic flux analysis indicated that these changes are driven by NADH and NADP+ in T. gondii infection. In sum, metabolic changes in host cells with T. gondii infection were similar during full infection, and kiss and spit. Autofluorescence lifetime imaging can non-invasively monitor metabolic changes in host cells over a microbial infection time-course. Many of these host changes induced by this protozoa parasite are like cancer cells metabolism.
Abstract:
The immune tumor microenvironment is widely implicated in brain tumor progression, bur yet how these cells are functionally connected, and which key pathways/signals are responsible for their tumor friendly status remain as a critical gap in knowledge and barrier for a successful clinical intervention. To first address this question, I investigate the role of the “CBM” (CARD–BCL10–MALT1 signalosome, a multiprotein signaling platform that control immune and inflammatory pathways via NF-κB/MAPK activation) impact on the brain tumor microenvironment. Recently studies suggested that tumor microenvironmental cells, which regulatory T (Treg) being particularly dependent on CBM, sustain their immune-suppressive functions via CBM activation. However, the brain tumor microenvironment is unique, and the majority of the non-neoplastic cells are tumor-associated macrophages (TAMs), either of peripheral origin or representing brain-intrinsic microglia, that create a supportive stroma for neoplastic cell expansion and invasion. The impact of CBM activation in these cells remained almost unknown until now. Using genetic knockout mice and immunocompetent orthotopic glioblastoma mouse models, I determine that CBM signaling in tumor associated macrophages also drives immunosuppression and tumor tolerance, thus positioning the pathway at the center of glioblastoma neuropathology. Importantly, I showed that a therapeutical strategy targeting the CBM using a BBB-penetrant MALT1 protease inhibitor abrogate tumor progression, increases mice survival and blocks pro-tumor “like-M2” macrophage formation/infiltration shedding light on the molecular basis of tumor microenvironment education of glioblastoma. These findings suggest that MALT1 may provide an effective approach for treating glioblastoma.
Abstract:
Primary myelofibrosis is a disease with a high unmet medical need since current treatments do not modify the natural history and are generally effective for 2-3 years. Myelofibrosis is characterized by a chronic inflammatory state with bone marrow fibrosis, resulting in ineffective hematopoiesis. Megakaryocytes are one of the main cellular drivers of myelofibrosis, exhibiting hyperproliferation and morphological abnormalities. However, the mechanisms by which inflammatory factors in the bone marrow environment contribute to fibrotic progression are not well understood. Our study revealed that the IL-13 signaling pathway contributes to the fibrotic progression in myelofibrosis by promoting megakaryopoiesis. IL-13-stimulated megakaryocytes express high levels of TGF-b on the cell surface, subsequently inducing the expression of fibrotic genes in bone marrow mesenchymal stromal cells. Importantly, we observed elevated levels of both IL-13 and its receptor in human plasma and bone marrow biopsies from myelofibrosis patients, underscoring the relevance of our findings in mice to human pathophysiology. Through in vivo modulation of IL-13 signaling, we demonstrated that IL-13 overexpression promotes disease progression, whereas reduction of IL-13/IL-4 signaling ameliorates several disease features, including fibrosis. Notably, we identified bone marrow T cells and mast cells as the sources of IL-13 production. Collectively, our study uncovered a previously uncharacterized signaling pathway implicated in the fibrotic progression of myelofibrosis, suggesting that its inhibition could potentially serve as a therapeutic strategy for treating this disease.
Abstract:
Long interspersed element-1 (LINE-1) is the only active, protein-coding transposon in humans that can self-propagate via RNA intermediates. LINE-1 overexpression and somatically-acquired LINE-1 copies are commonly detected in human cancers with TP53 mutations. A recent pan-cancer analysis found associations between somatically-acquired LINE-1 insertions and chromosomal rearrangements, suggesting that LINE-1 retrotransposition activity may represent a major source of chromosomal instability in cancer genomes. To address this hypothesis, we have developed a TetOn system to induce LINE-1 expression in TP53 deficient retinal pigment epithelial (RPE-1) cells. Using this system, we have performed whole genome sequencing (WGS) of single cells or clonal outgrowths to comprehensively assay the genomic alterations generated after induction of LINE-1 expression. We have found that clonal outgrowths induced with LINE-1 expression contain one or multiple copy-number alterations, including copy-number losses and gains, and whole chromosome losses. We have also detected a variety of short and long-range genomic rearrangements with one or multiple breakpoints attributed to LINE-1-encoded ORF2p endonuclease, demonstrating a direct role of LINE-1 in creating large structural rearrangements. Strikingly, we have found that cell exposed to LINE-1 expression also contain complex rearrangements such as chromothriptic chromosomes, suggesting that LINE-1 can elicit chromothripsis. In addition, we are characterizing the DNA damage induced by LINE-1 retrotransposition and have found that LINE-1 expression activates the DNA damage response (e.g., ATM targets including gH2AX, phosphorylated KAP1 and phosphorylated RAD50) and increases the frequency of cells with abnormal nuclear structures such as micronuclei. These studies are significant for providing insights into the scope of LINE-1-mediated genome instability and should inform our efforts to exploit LINE-1 genotoxicity as a cancer therapeutic strategy.
Abstract:
Animal gametes typically develop through some or all of gametogenesis as syncytia of cells attached by stable intercellular bridges called ring canals. Ring canals are important conduits allowing the movement of mRNAs, proteins, and organelles between newly synthesized sibling cells. Despite the conservation of germline ring canals across evolution, how canonical cytokinesis is altered during germ cell division to produce ring canals is poorly understood. Using time-lapse imaging of cytokinesis proteins in dividing germ cells in the Drosophila male germline, we observe that ring canal formation occurs via reorganization of the germline midbody, a structure classically associated with its function in recruiting abscission-regulating proteins in complete cytokinesis. However, unlike canonical midbodies, the germ cell midbody is short-lived, and rapidly reorganizes to join the proteins in the midbody ring resulting in a ring canal with an open lumen. This reorganization from midbody-to-ring canal is accompanied by a ~5-fold decrease in midbody protein fluorescence intensity suggesting that degradation or relocalization is required for ring canal formation. We found a remarkably similar midbody-to-ring canal transformation in mouse and Hydra testes, suggesting it is an intrinsic feature of gamete formation. We also identify a component necessary for germ cell midbody reorganization, the conserved mitotic kinase called Citron kinase/Drosophila Sticky. To identify additional ring canal promoting factors, we performed a candidate RNAi screen targeting mitotic kinases and phosphatases, as well as enzymes with important roles in ubiquitin homeostasis. We have identified several genes, some of which are uncharacterized, that are required for proper ring canal architecture and size suggesting an important role for post-translational modifications of germ cell midbody proteins in mediating midbody reorganization during ring canal biogenesis.
Abstract:
A deep understanding of viral infection mechanisms is essential for the development of effective treatments, the design of new drugs targets, and the identification of promising vaccine candidates. We characterized the dynamics of HIV-1 infection in vitro by simultaneously detecting viral RNA and viral DNA at the single-cell level. I used an in situ hybridization and fluorescence microscopy-based system to label HIV-1 nucleic acids simultaneously with viral and host proteins at a single-cell level. This allowed us to understand the HIV-1 life cycle events, including time course of viral entry, reverse transcription, integration with consecutive new spliced and unspliced HIV-1 transcripts, the detection of antisense RNA in the presence of integrase inhibitors, viral protein synthesis using different cells, including relevant clinical primary cells such as CD4+ lymphocytes and macrophages. The ability to visualize these nucleic acid intermediates in the context of viral or host proteins advance efforts to elucidate mechanisms of antiviral inhibition by small molecules or host restriction factors, enhance our understanding of latency reactivation, and further efforts for novel drug development.
We studied SARS-CoV-2 cellular tropism in human airway cell lines. We analyzed a panel of airway cell lines with various levels of ACE2 expression to identify models of SARS-CoV-2 infection. We found that the H522 human lung adenocarcinoma cells were naturally permissive to SARS-CoV-2 infection despite undetectable expression of ACE2. We confirmed that SARS-CoV-2 replication is indeed completely independent of ACE2 in H522 cells but dependent on heparan sulfates and the E484D substitution within the Spike. Furthermore, we showed that a subset of the ACE2 positive non-permissive cell lines express high basal levels of interferon-stimulated genes, and this mechanism is mediated by the cGAS-STING sensing pathway. These findings suggest that SARS-CoV-2 replication can proceed in complete absence of ACE2 and that the innate immunity is a key determinant of SARS-CoV-2 cellular tropism. These findings may explain the complex SARS-CoV-2 pathogenesis in vivo as it shows that factors independent of ACE2 can define cellular tropism.
Abstract:
Metabolic adaptation to rapid changes in the cellular microenvironment is essential for survival. Oxidative stress response (OSR) is a key component of metabolism that maintains cellular homeostasis by protecting cells from damaging molecules. While the ability to maintain homeostasis is beneficial in healthy cells, OSR is co-opted in disease sates such as obesity and cancer. Increased metabolic demand and cellular proliferation in disease states are associated with dysregulation of cellular signaling networks as well as increased OSR due to accumulation of damaging reactive oxygen/nitrogen species (ROS/RNS). The signaling-mediated regulation of OSR is an intricate and complex process that plays an important role in coordinating mechanism of cellular detoxification and survival. Moreover, rapid stress response, in the seconds-to-minute timeframe, relies on cellular signaling networks mediated by protein post-translational modifications (PTMs) such as phosphorylation. To identify the dynamic regulatory networks of OSR we annotated the published tyrosine phosphoproteome (pY) with a particular focus on metabolic enzymes. We found that enzymes involved in fatty acid and lipid metabolism linked to OSR pathways were enriched in the published datasets. Therefore, we interrogated OSR in a high fat diet (HFD)-induced obesity mouse model by employing parallel quantitative mass spectrometry-based pY phosphoproteomics and polar metabolomics. We map the concerted additive effects of pY on enzymes and corresponding changes in metabolites using our systems level approach. By characterizing HFD altered metabolic and signaling networks, we demonstrate that HFD rewires nucleotide and glutathione metabolism in a sex specific manner. We further validate functional role of pY on select enzymes using kinetic enzyme assays. Our work illustrates that by defining the effect of phosphorylation on enzyme kinetics, we identify the convergence zone where cellular signaling ‘tunes’ metabolic adaptation and OSR in obesity.
Abstract:
The central nervous system (CNS) has classically been considered “immune-privileged”. However, emerging viruses with the capacity to enter the CNS have challenged what we consider immune privilege. Specifically, neuropathogenic Enterovirus-D68 (EV-D68) strains have been associated with a polio-like paralysis called acute flaccid myelitis (AFM) in some children. AFM remains a rare infection outcome, suggesting that host immune responses may limit neurological disease. Studies of such immune responses remain poorly understood. Human stem cell-derived cerebral organoids have emerged as three-dimensional in vitro systems, displaying the heterogeneity of cell types present in our brains. Thus, to better dissect differences between non-neuropathogenic and neuropathogenic EV-D68 strains, I generated and infected early and late cerebral organoids. I found that cerebral organoids were more permissive to neuropathogenic EV-D68 infection compared to non-neuropathogenic EV-D68 in early organoids. Surprisingly, non-neuropathogenic EV-D68 replicated to the level of neuropathogenic EV-D68 in late organoids. Neural rosettes, accumulated hubs of neural progenitors, are highly expressed in early organoids and decrease as organoids age. Interestingly, these neural rosettes were not infected with EV-D68, suggesting that they may be immune signaling hubs, limiting virus infection. These data suggest that controlling immune responses may limit neuropathogenesis and also highlight the utility of using cerebral organoids to examine host immune responses to neurotropic virus infection.