Browsing by Subject "Host-Pathogen Interactions"
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Item The Art of Viral Oncogenesis: Lessons from Human Papillomavirus and Polyomavirus Transformed Cancers(2020-05-01T05:00:00.000Z) Zhao, Jiawei; Xu, Jian; Banaszynski, Laura; Pfeiffer, Julie K.; Wang, RichardViruses account for about 15% of all human cancer. Understanding viral oncogenesis can substantially broaden our general knowledge on the molecular mechanisms of carcinogenesis. In this dissertation, I focused on two types of DNA oncoviruses, human papillomavirus (HPV) and polyomavirus (HPyV), and identified novel mechanisms by which these two types of viruses cause human cancers. In HPV transformed cancer cells, I identified a novel circular RNA species, circE7, that spans and encodes the HPV E7 oncoprotein. I later demonstrated that circE7 translated E7 protein accounts for a substantial proportion of the E7 protein in a HPV transformed cancer cell line, and whose absence significantly impacts cancer cell proliferation in vitro and in vivo. In Merkel Cell Polyomavirus (MCPyV) transformed MCC cancer cells, I identified the activation of non-canonical NF-κB pathway activation by the MCPyV small T (ST) oncoprotein. I further demonstrated that the ectopically activated non-canonical NF-κB pathway is required for cell growth in low serum. The inhibition of non-canonical NF-κB signaling by a small peptide inhibitor also resulted in impaired cell growth in vitro and in vivo due to ER stress mediated apoptosis, suggesting a novel therapeutic intervention strategy for viral positive MCC (VP-CC) patients.Item Autophagy in Antiviral Immunity(2012-08-15) Orvedahl, Anthony Walter; Levine, BethAutophagy is an evolutionarily conserved pathway in which cytoplasmic material is sequestered in a double-membrane vesicle and delivered to the lysosome for degradation. During times of stress, autophagy functions to generate essential nutrients through the degradation of non-essential cytoplasmic contents. It is also the only known mechanism for removal of damaged or superfluous organelles and cytoplasmic contents that are too large to be degraded by the proteasome. Given the critical role for autophagy in stress response and in maintaining cell cytoplasmic quality control, it is not surprising that autophagy plays an essential role in the host response to infection, and that microbes have evolved mechanisms to counteract or evade autophagy. In this work, we studied the role of autophagy inhibition in a mouse model of herpes simplex virus type I (HSV-1) encephalitis, investigated the role of autophagy in protection against Sindbis virus infection of the central nervous system, and identified novel host genes involved in targeting viral proteins to the autophagy pathway. We found that the HSV-1 encoded neurovirulence protein ICP34.5 interacted with the host autophagy protein Beclin 1, and that this interaction was essential for HSV-1 neurovirulence. This was the first example of a viral virulence protein that targets host autophagy, and provided evidence that autophagy functions in innate immunity to viruses. In the second study, we found that the host autophagy gene Atg5 was required to protect against lethal Sindbis virus CNS diseases, and that autophagy targeted viral proteins for degradation in brains of infected mice and cells in vitro. We found that the autophagy adaptor protein p62 was involved in targeting viral proteins for autophagic degradation and this promoted survival of infected cells. This study demonstrated that clearance of viral proteins by autophagy was an important mechanism for cellular and organismal survival during viral infection. Lastly, we performed a genome-wide siRNA screen to identify novel host factors required for autophagic targeting of viral proteins. We identified previously unappreciated cellular networks and genes that were involved in targeting viral proteins for autophagy. One of these factors, SMURF1, is an E3 ubiquitin ligase that not only functions to target viral proteins, but is also involved in targeting damaged mitochondria for autophagic clearance.Item Biochemical Characterization of IpaH E3 Ubiquitin Ligase Effector Proteins and Their Host Substrates(August 2021) Hansen, Justin Mark; Orth, Kim; Sperandio, Vanessa; Reese, Michael L.; Alto, NealShigella flexneri is a gram negative pathogen that utilizes its type 3 secretion system (T3SS) to inject effector proteins in the cytoplasm of host cells to manipulate host cells processes. T3SS effectors are able to post translationally modify host proteins to reprogram intracellular signaling pathways, actin dynamics, membrane trafficking, and innate immune pathways. This allows Shigella to modify the intracellular environment to be conducive to bacterial replication and dissemination to neighboring cells. Shigella flexneri and other bacteria including Salmonella and Yersinia secreted E3 ubiquitin ligases into the host cell cytoplasm via the Type III secretion system (T3SS) apparatus. The invasion plasmid antigen Hs (IpaHs) are a novel family of bacterial E3 ubiquitin ligases that are secreted by Shigella, Salmonella, and Yersinia. These bacterial enzymes highjack the host ubiquitin conjugation machinery by binding to ubiquitin-charged E2 conjugating enzymes and facilitating direct transfer of ubiquitin onto host substrates. IpaH effectors induce polyubiquitination and subsequent proteasomal degradation of their substrates during bacterial infection. The effector substrate interaction of IpaH1.4/2/5 and HOIP was previously characterized. I went on to identify that IpaH2.5 is able to inhibit the in vitro catalytic activity of HOIP via mono-ubiquitination of catalytic lysine residues in the HOIP ring-between-ring domain (RBR-C). Subsequent to this Ubiquitin activated interactive trapping (UBAIT) screening was then utilized to identify the host substrate of IpaH7.8, Gasdermin B (GSDMB). GSDMB belongs to a large family of pore forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to inflammatory disease, its function in human physiology remains poorly understood. I investigated a previously unrecognized host-pathogen conflict between GSDMB and the IpaH7.8 effector protein encoded by Shigella flexneri. Through extensive biochemical and cellular characterization, I show that IpaH7.8 ubiquitinates and targets GSDMB for proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of Natural Killer cells by suppressing Granzyme-A mediated activation of GSDMB. In contrast to the canonical function of most Gasdermin-family members, GSDMB does not inhibit Shigella by lysing infected cells. Rather, GSDMB exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveals a mechanism employed by pathogens to counteract this host defense system.Item The Biological Role of Stochasticity from Molecules to Communities(2013-07-17) Kittisopikul, Mark Andrew; Ranganathan, Rama; Süel, Gürol M.; Alto, Neal; Rizo-Rey, JoséThe presence of stochasticity in biology engenders the question of the role of such randomness. While stochasticity may be an artifact of biochemical interactions, it could also be an actively regulated aspect of life. Here I investigate how noise may be encoded within the molecular interactions of a biochemical network and then examine how those consequences propagate to higher levels of organization including isogenic populations of cells and host-pathogen interactions. I particularly look into how this variability impacts the ability of populations to respond to their environments. I conclude that stochasticity is selectable property of life that adds robustness to biological processes during periods of uncertainty.Item Characterization of Host Factors Affecting Viral Entry(2019-04-10) Rinkenberger, Nicholas Ryan; Alto, Neal; Schoggins, John W.; Pfeiffer, Julie K.; Reese, Tiffany A.Viruses are obligate, intracellular parasites. For a virus to infect a host cell, it must gain access to the interior of the host cell by some means. In animals, this often involves the exploitation of host processes such as receptor-mediated endocytosis and vesicular trafficking. Zika virus is an emerging arbovirus with global health and economic impacts. Interestingly, while Asian lineage Zika virus causes human disease and has been associated with severe neurological complications, African lineage Zika virus has only rarely been reported to cause human disease. Large strides have been made in understanding Zika virus infection. However, the mechanism used by Zika virus to enter host cells remains somewhat obscure. In chapter 2, I delineate and compare the pathway utilized by both Asian and African lineage Zika virus to enter host cells. I find that these viruses require clathrin-mediated endocytosis and Rab5a function in a conserved manner. Additionally, all Zika virus strains tested were sensitive to pH in the range of 6.5-6.1 and were reliant on endosomal acidification for infection. I found that Zika virus preferentially fuses with late endosomes. Comparing lineages, Zika virus enters cells in a highly conserved manner. Just as viruses have evolved to exploit host factors to promote their entry and replication, hosts have developed mechanisms of defense against viral infection. Recognition of viral infection by vertebrate hosts results in the expression and secretion of interferon. Interferon signaling subsequently results in the induction of hundreds of interferon-stimulated genes (ISGs) which restrict pathogen infection. Some of these ISGs specifically block viral entry. Surprisingly, a small group of ISGs was previously identified which actually promote viral infection. In chapter 3, I characterize the mechanism of action of MCOLN2, one of the ISGs found to promote viral infection. I assign a role for MCOLN2 in modulating viral entry. I show that MCOLN2 specifically promotes viral vesicular trafficking and subsequent escape from endosomal compartments. This mechanism requires channel activity, occurs independently of antiviral signaling, and broadly applies to enveloped RNA viruses that require endosomal acidification for infection, including influenza A virus, yellow fever virus, and Zika virus.Item Identification of an Interferon Signaling Pathway Linking Membrane Cholesterol Accessibility to Host Defense Against Pathogens(2020-08-01T05:00:00.000Z) Abrams, Michael Edward; Shiloh, Michael; Alto, Neal; Radhakrishnan, Arun; Schoggins, John W.Interferon-γ (IFN-γ) is a multipotent cytokine that is critical to the host innate immune defense against bacterial infection, and functions through transcriptional induction of hundreds of IFN-γ stimulated genes (γ-ISGs). However, the antibacterial roles of many γ-ISGs remain poorly defined. Here, I describe my efforts to characterize mechanisms by which specific γ-ISGs confer cell-intrinsic immunity against bacterial pathogens. Unexpectedly, I found that IFN-γ-activated macrophages secreted a soluble product that potently inhibited infection of the Gram-positive intracellular pathogen Listeria monocytogenes. To identify this factor, I first created a cDNA lentiviral library of more than 400 highly representative γ-ISGs and carried out a gain-of-function flow cytometry screen to determine the effect of each gene on infection. The results of this screen identified eight genes that potently reduce infection. Next, I determined whether these γ-ISGs produced a soluble molecule that could suppress L. monocytogenes infection in trans. Notably, conditioned media from Cholesterol 25-Hydroxylase (CH25H)-expressing cells potently enhanced bacterial resistance of naive cells, suggesting it may be responsible for the previously observed activity in macrophages. Indeed, Ch25h-/- macrophages failed to produce a soluble antibacterial metabolite, while the enzymatic product of CH25H, 25-Hydroxycholesterol (25HC), potently inhibited L. monocytogenes infection. I demonstrated that 25HC inhibits infection of non-phagocytic cells from diverse tissue lineages, and that administration of 25HC to mice significantly reduced bacterial burden in an oral gavage model. Furthermore, I found that 25HC blocks L. monocytogenes cell-to-cell spread by attenuating the formation of plasma membrane protrusions. In collaboration with the post-doctoral fellow Kristen Johnson, I have used toxin-based biosensors to determine that activation of acyl-CoA: cholesterol acyltransferase (ACAT) rapidly mobilized a specific pool of cholesterol termed “accessible cholesterol” from the plasma membrane (PM) to the ER. Importantly, the antibacterial function of 25HC was dependent on this ability to deplete PM accessible cholesterol. Together, these studies have uncovered a heretofore unknown mechanism by which IFN-mediated reorganization of the PM restricts dissemination of intracellular pathogens.Item Identification of Receptor Transporting Proteins As Conserved Antiviral Effectors in Vertebrates(2021-05-27) Boys, Ian Nicholas; Shiloh, Michael; Alto, Neal; Yan, Nan; Hancks, Dustin C.; Schoggins, John W.Viruses and their hosts are engaged in "genetic arms races" in which each side attempts to gain the advantage over evolutionary time. Results of these conflicts are wide-ranging: viruses diversify, hosts establish species-specific barriers to some viruses while remaining susceptible to others, and the lines for future genetic conflicts are drawn. In mammals, many antiviral effectors -- proteins that directly inhibit viral infection -- show species- or lineage-specific properties which are believed to be the result of past or ongoing conflicts. Bats harbor a greater diversity of viruses than any other mammalian order, and a growing body of research has described unique adaptations in bats that are in part responsible for, and perhaps a response to, this unique status. We hypothesized that the frequent encounters between bats and viruses would drive unique adaptations in the antiviral effectors that serve on the front lines of virus-host genetic conflicts. We identified RTP4 from the bat Pteropus alecto as a potent inhibitor of flavivirus infection. Mechanistic studies determined that RTP4 is an RNA-binding protein that associates with flavivirus replication machinery, binds replicating viral RNA, and suppresses viral genome amplification. Phylogenomic analysis revealed that RTP4 has evolved under positive selection in several mammalian lineages, consistent with a model in which host-virus conflicts have shaped its evolution as a restriction factor not only in bats but across mammals. We assessed the antiviral efficacy of diverse mammalian RTP4 orthologs and found that orthologs exhibit striking patterns of antiviral specificity. Further highlighting the specificity of the host-virus arms race, experimental evolution demonstrated that a flavivirus can mutate to escape RTP4-imposed restriction in a species-specific manner. In follow-up work, we identified signatures of positive selection in several non-mammalian RTP homologs, indicative of a putative role in innate immunity. We screened a collection of vertebrate RTPs against a panel of viruses and identified antiviral RTPs in the African clawed frog, Xenopus laevis. These antiviral Xenopus RTPs exhibit mosaic phenotypes that resemble those of mammalian RTP4 orthologs. Within the context of our findings with mammalian RTP4, these data suggest that Receptor Transporter Proteins are involved in host-virus genetic conflicts outside of Mammalia.Item Inhibition of Cell Proliferation through Regulated Intramembrane Proteolysis of CREB3L1(2012-07-10) Denard, Bray Standard; Ye, JinCREB3L1/OASIS is a cellular transcription factor synthesized as a membrane- bound precursor and activated by regulated intramembrane proteolysis in response to stimuli like ER stress. By comparing gene expression between Huh7 subclones that are permissive for hepatitis C virus (HCV) replication versus the non-permissive parental Huh7 cells, we identified CREB3L1 as a host factor that inhibits proliferation of virus-infected cells. Upon infection with diverse DNA and RNA viruses, including murine gamma-herpesvirus 68, HCV, West Nile virus (WNV), and Sendai virus, CREB3L1 was proteolytically cleaved, allowing its NH2 terminus to enter the nucleus and induce multiple genes encoding inhibitors of the cell cycle to block cell proliferation. Consistent with this, we observed a necessity for CREB3L1 expression to be silenced in proliferating cells that harbor replicons of HCV or WNV. Our results indicate that CREB3L1 may play an important role in limiting virus spread by inhibiting proliferation of virus-infected cells. Doxorubicin is used extensively for chemotherapy of diverse types of cancer, yet the mechanism through which it inhibits proliferation of cancer cells remains unclear. Here we report that doxorubicin stimulates de novo synthesis of ceramide, which in turn activates CREB3L1, a transcription factor synthesized as a membrane-bound precursor. Doxorubicin stimulates proteolytic cleavage of CREB3L1 by Site-1 Protease and Site-2 Protease, allowing the NH2-terminal domain of CREB3L1 to enter the nucleus where it activates transcription of genes encoding inhibitors of the cell cycle, including p21. Knockdown of CREB3L1 mRNA in human hepatoma Huh7 cells and immortalized human fibroblast SV589 cells conferred increased resistance to doxorubicin, whereas overexpression of CREB3L1 in human breast cancer MCF-7 cells markedly enhanced the sensitivity of these cells to doxorubicin. These results suggest that measurement of CREB3L1 expression may be a useful biomarker in identifying cancer cells sensitive to doxorubicin.Item The Interferon Stimulated Gene Product Lymphocyte Antigen 6 Complex, Locus E Promotes Entry of a Subset of Diverse RNA Viruses and Inhibits Infection by Coronaviruses(2019-02-05) Mar, Katrina Bockying; Hooper, Lora V.; Alto, Neal; Schmid, Sandra; Schoggins, John W.Interferons (IFNs) contribute to cell-intrinsic antiviral immunity by inducing hundreds of IFN-stimulated genes (ISG). In a screen to identify novel antiviral factors, the Schoggins lab unexpectedly uncovered a subset of genes that enhanced viral infection. Here, I describe my personal efforts to study lymphocyte antigen 6, locus E (LY6E), a protein which was identified in the screen to enhance the infection of viruses from Flaviviridae, Orthomyxoviridae, Retroviridae, and Alphaviridae viral families. In my studies, I confirmed that LY6E promotes viral infection of viruses from the same families, as demonstrated by both ectopic overexpression and endogenous knockout approaches. Using influenza A virus (IAV) as a model, I narrowed the enhancing effect of LY6E specifically to the entry step of uncoating, which precedes release of the viral genome into the cytoplasm and is required for viral replication. I also observed that the viral enhancement phenotype is conserved across evolution, as orthologs from bat, rhesus macaque, and mouse exerted a similar effect. To understand the physiological relevance of viral enhancement at the cellular level, I generated Ly6e conditional knockout mice and crossed them to multiple Cre recombinase transgenic mouse strains. As a result, I obtained mice with specific ablation of Ly6e in distinct immune cell compartments. From both ex vivo and in vivo studies using the Ly6e knockout mice, I concluded that Ly6e in alveolar macrophages is important for optimal defense against IAV infection. Finally, in collaboration with the postdoctoral fellow Stephanie Pfaender, I have shown that LY6E also possesses potent antiviral activity against a distinct subset of enveloped RNA viruses. Cumulatively, my work has uncovered three unique ways by which the ISG LY6E may contribute to the antiviral immune response. This work also provides insight regarding the multi-faceted ways a single ISG can provide broad protection against infection by viruses from diverse viral families.Item Interplay Between Tryptophan Metabolites and the Virulence Factors of Enteric Pathogens(2020-08-01T05:00:00.000Z) Kumar, Aman; Winter, Sebastian E.; Sperandio, Vanessa; Conrad, Nicholas; Tu, BenjaminThe human gut consists of a complex milieu of several small molecules that helps in shaping its overall chemistry and biogeography. Trillions of bacteria, collectively known as the gut microbiota, colonize this landscape and occupy a specific niche. Small molecules derived from diet, gut microbial metabolism, and host metabolic activity have an impact on dictating the underlying gut microbiome composition. Gut bacterial populations effectively sense these molecular signatures and modulate gene expression to colonize this niche. An invading intestinal pathogen, to effectively colonize the gut, must sense and respond to the molecular signatures in the gut, which leads to effective colonization and infection. Diet is the principal source of energy by the intake of three main nutrients: carbohydrates, proteins, and fats. Tryptophan is an essential amino acid taken from the diet and plays an important role in protein biosynthesis. Tryptophan is also metabolized to different small molecules and its metabolic products are known to be present in high concentrations in the body. Upon ingestion, tryptophan is readily available in the luminal environment of the gut. Abundant levels of tryptophan are also absorbed by intestinal epithelial cells and is made available in different cellular compartments for normal physiological processes. Tryptophan present in the gut lumen can be further metabolized to numerous small molecules by the action of the gut microbial metabolic activity. Indole is one of the most abundant tryptophan-derived metabolites present in the gut and is absorbed by the host epithelial cells. The host can also metabolize tryptophan to various small molecules including serotonin that is made available in the gut lumen upon release from the host cells. Bacteria in the gut sense these bacterial and host-derived small molecules to colonize and maintain their niche. Similarly, an invading food-borne pathogen such as Enterohemorrhagic Escherichia coli (EHEC), which causes gastroenteritis by primarily colonizing the human colon, sense these small molecules and respond in a way that is conducive for its colonization and virulence gene expression. The role of these highly abundant tryptophan-derived small molecules in dictating the infectivity of an enteric pathogen remains unknown. Because the concentrations of these small molecules naturally present in the body are in the range of the drug concentrations that are used to treat certain diseases, it is possible to repurpose the information gained from these studies to treat intestinal infections. In the present study, we focused on two highly abundant tryptophan-derived small molecules present in the gut. Indole is derived from bacterial metabolism while serotonin is present via the action of host metabolism of tryptophan. Indole and serotonin are structurally similar and therefore may have similar effects against a pathogen in vivo. Indeed, we identified that both indole and serotonin decrease the virulence of the human pathogen Enterohemorrhagic E. coli (EHEC) and the mouse pathogen Citrobacter rodentium. We used multiple strategies including genetic manipulation, pharmacological inhibitors, and knock-out murine models to show that both indole and serotonin are inhibitory signals for virulence gene expression in EHEC and C. rodentium. We further investigated the mechanism used by these pathogens to sense these signals. We identified the first bacterial receptor for both indole and serotonin, and showed that these signals are sensed by a bacterial membrane bound histidine kinase CpxA. Upon sensing indole or serotonin, CpxA dephosphorylates itself and the transcription factor CpxR. In its phosphorylated state, CpxR directly activates expression of the virulence genes, its dephosphorylation prevents its action, decreasing expression of these genes. Together, our studies highlight the importance of sensing small molecules and understanding the gut biogeography by invading pathogens to successfully colonize the gut.Item Localization and Function of Bacterial Type III and IV Effector Proteins(2017-07-31) Jimenez, Alyssa; Winter, Sebastian E.; Alto, Neal; Sperandio, Vanessa; Schoggins, John W.Eukaryotic cell signal transduction networks are highly dynamic and complex systems largely composed of signaling enzymes with modular protein interaction domains and subcellular localization motifs. These sophisticated regulatory mechanisms are crucial to the fidelity and efficacy of information relay in both space and time. Bacterial effector proteins are virulence factors that are directly secreted from the bacteria into the host cytosol and function to rewire eukaryotic signaling networks to establish an environment for bacterial survival. While much effort has gone into substrate identification and biochemical characterization of bacterial effector proteins, it remains unclear how these bacterial enzymes are able to amplify their signaling events to efficiently usurp the robust signaling networks of eukaryotic cells. To this end, I utilized a yeast genetic screen to ask whether bacterial effectors proteins are able to interact with eukaryotic membranes, structures that serve as organizational platforms for the assembly of multi-protein complexes critical for eukaryotic signal transduction. By focusing on a family of bacterial guanine nucleotide exchange factors (GEFs) that activate Rho-family GTPases, and are indispensable for the characteristic accumulation of actin at the site of bacterial attachment and invasion, I have identified a membrane-localization domain in the Salmonella effectors SopE and SopE2 that regulates the ability of these effectors to activate Rho GTPases and invade eukaryotic cells. This membrane-localization domain may function to concentrate these effectors to the highly curved membranes found during Salmonella invasion. Additionally, a polybasic domain identified in the Shigella GEF IpgB1 was found to spatially and temporally regulate actin dynamics. Furthermore, the subcellular location of previously uncharacterized Legionella effectors were found to localize to the vacuoles in yeast and may play a role in regulating vacuolar fusion. Lastly, to gain a greater comprehension of the complex interplay of Salmonella SPI-2 effectors, I developed a collection of Salmonella mutant strains consisting of single SPI-2 effector deletions and a series of combinatorial mutants that are deficient in 2 to 29 effectors. This study expands our knowledge on fundamental processes critical to host-pathogen interactions and provides important mechanistic insights into the spatiotemporal regulation of bacterial effector proteins.Item Mechanisms of Macrophage Detection and Control of Mycobacterium tuberculosis Infection(2016-04-15) Collins, Angela Christine; Wakeland, Edward K.; Shiloh, Michael; Hansen, Eric J.; Marciano, Denise; Russell, David W.; Yarovinsky, FelixMacrophages use different mechanisms to recognize and respond to Mycobacterium tuberculosis infection. Macrophage recognition of M. tuberculosis is characterized by the production of a robust type I interferon response dependent on the activation of a cytosolic surveillance pathway by the recognition of M. tuberculosis DNA in the cytosol. The DNA sensor recognizing M. tuberculosis DNA and initiating activation of the cytosolic surveillance pathway has yet to be defined. Here we describe a role for the recently characterized DNA sensor cGAS in the detection of M. tuberculosis infection and initiation of the type I interferon response as well as a role for cGAS in the targeting of M. tuberculosis to the autophagosome. We demonstrate that cGAS deficiency is associated with decreased survival in a mouse model of M. tuberculosis infection. The second part of this thesis explores how macrophages respond to M. tuberculosis infection. We previously showed that in mice M. tuberculosis infection induces the expression of the carbon monoxide producing enzyme heme oxygenase (HO1) in the macrophage and that the CO is sensed by M. tuberculosis to initiate a dormancy program. Mice deficient in HO1 succumb to M. tuberculosis infection more readily than wild-type (WT) mice. While the mechanisms used by mouse macrophages to control intracellular M. tuberculosis infection, including nitric oxide synthase, the respiratory burst, acidification and HO1 are well studied, how human macrophages control M. tuberculosis infection is less well understood. Here we show that HO1 is induced by and colocalizes with M. tuberculosis in both mouse and human tuberculosis lesions, and that M. tuberculosis induces and colocalizes with HO1 during human macrophage infection in vitro. HO1 enzymatic activity in human macrophages is necessary for inflammatory cytokine production and for control of intracellular M. tuberculosis replication. Finally, we find that a polymorphism in the HO1 promoter is associated with susceptibility to human tuberculosis. Thus, we demonstrate an important role for HO1 in controlling human tuberculosis.Item Mycobacterium Tuberculosis Virulence Factor Mpt64 Targets the Endoplasmic Reticulum(2019-04-10) Stamm, Chelsea Elizabeth; Sperandio, Vanessa; Shiloh, Michael; Alto, Neal; Winter, Sebastian E.Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that M. tuberculosis can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, M. tuberculosis is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secret virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. I hypothesized that M. tuberculosis secreted proteins that target host membranes are vital for M. tuberculosis to adapt to and manipulate the host environment for survival. Thus, I characterized nearly 200 secreted proteins from M. tuberculosis for their ability to associate with eukaryotic membranes using a live-dead, temperature sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. I identified five M. tuberculosis secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during M. tuberculosis infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in M. tuberculosis pathogenesis and provide a basis for further investigation into their molecular mechanisms.Item Neutrophil-Derived IFN-γ in Toxoplasma gondii Infection and Innate Immunity(2014-11-19) Sturge, Carolyn Rowena; van Oers, Nicolai S. C.; Yuan, Dorothy; Zhang, Chengcheng "Alec"; Koh, Andrew Y.; Yarovinsky, FelixInterferon-gamma (IFN-γ) is a major cytokine that is critical for host resistance to a broad range of intracellular pathogens. Production of IFN-γ by Natural Killer (NK) and T cells is initiated by the recognition of pathogens through Toll-like receptors (TLRs). In an experimental model of toxoplasmosis we have identified the presence of a non-lymphoid source of IFN-γ that was particularly evident in the absence of TLR-mediated recognition of Toxoplasma gondii. Flow-cytometry and morphological examinations of non-NK/non-T IFN-γ-positive cells identified neutrophils as the cell type capable of producing IFN-γ. Selective elimination of neutrophils in TLR11-/- mice infected with the parasite resulted in acute susceptibility similar to that observed in IFN-γ-deficient mice. These data show that neutrophils are a biologically significant source of IFN-γ during T. gondii infection. Additionally, we investigated the role of neutrophil IFN-γ in another intracellular infection, Salmonella typhimurium, and found that neutrophils were also IFN-γ-positive. Examination of neutrophils in different locations in a mouse model revealed that they all expressed low amounts of IFN-γ regardless of infection status. In particular, the bone marrow niche contained an IFN-γ+ population that was negative for the Ly-6G marker characteristic of mature neutrophils in peripheral tissues. Recent work defining neutrophil developmental stages by flow-cytometry allowed us to discern that precursor neutrophils at the promyelocyte stage (Ly-6G negative) were positive for IFN-γ. Furthermore, neutrophil-derived IFN-γ was prestored in granules during neutrophil lineage development although the mechanisms behind this phenomena are not yet understood. This work, combined with the recent work of other laboratories, suggests that neutrophils can have defined phenotypes and cytokine production similar to that of T cells or Innate Lymphoid cells (ILCs). These findings have broad implications for all disease states where neutrophils are the first responders to infections.Item The Pivotal Role of cGAS in Host Defense Against Mycobacterium Tuberculosis and in Particle-Induced Immunity(2017-11-02) Cai, Haocheng; Fu, Yang-Xin; Hooper, Lora V.; Yan, Nan; Chen, Zhijian J.The cGAS-cGAMP-STING pathway is a major DNA sensing pathway in the cytosol. The DNA sensing mechanism is crucial for host defense against bacteria, viruses, and parasites. In the previous study from our lab, the cGAS-STING pathway has been identified as being critical for antiviral activities. However, anti-bacterial activity is more complicated due to the complexity of the invading agents. Mycobacterium tuberculosis (Mtb) is a pandemic pathogen that triggers millions of deaths every year. It is discovered that Mtb ejects its DNA component into host cytosol. We have uncovered the cGAS pathway, which elicits type I interferons and autophagy in macrophages and other cell types, as a defending mechanism against Mtb infection. cGAS is critical for mouse survival against chronic Mtb infection. In conclusion, cGAS is critical for the host defense against Mtb, especially in the induction of type I interferon and autophagy. Previous studies revealed that some fusogenic liposomes and enveloped virus-like particles can activate type I interferons. Here we have shown that fusogenic liposomes and nonenveloped virus-like particles can activate the cGAS pathway and trigger interferon production, which is associated with the amount of cytosolic DNA. Moreover, cGAS and STING are critical for the humoral immune response elicited by virus-like particles. Overall, we have delineated the significant role of cGAS in mediating the immune response induced by fusogenic liposomes and virus-like particles. Additionally, we have proven that cGAS heterozygous mice have largely retained the antiviral activity in herpes simplex virus infection, compared with wildtype mice. This has shed light on the development of cGAS inhibition therapies against autoimmune diseases. Overall, this dissertation has identified the pivotal roles of the cGAS-STING DNA sensing pathway in Mycobacterium tuberculosis infection and particle-induced immunity, as well as the effect of cGAS heterozygosity on antiviral activities.Item Regulation of Expression and Regulated Intramembrane Proteolysis of CREB3L1(2013-07-26) Chen, Qiuyue; Liang, Guosheng; Chen, Zhijian J.; Pfeiffer, Julie K.; Jain, MamtacAMP response element binding protein 3-like 1 (CREB3L1) is a transcription factor synthesized as a membrane-bound precursor and activated through Regulated Intramembrane Proteolysis (RIP). Previous study has shown that CREB3L1 was proteolytically cleaved upon viral infection, allowing its NH2-terminus to enter nucleus to drive genes encoding inhibitors of the cell cycles. As a result, CREB3L1 inhibited replication of cells infected by some virus including Hepatitis C virus (HCV). HCV does not replicate efficiently in wild-type human hepatoma Huh-7 cells, but it replicates robustly in certain subclones of Huh-7 cells. It is found that the expression of CREB3L1 is blocked in the cells permissive for HCV replication. However, how CREB3L1 is silenced in those cells remains unknown. Here we showed that CREB3L1 and other anti-viral genes like myxovirus resistant 1 (MX1) were epigenetically silenced through DNA methylation in different subclones that are permissive for HCV replication. Methylation microarray analysis suggested that Huh-7 cells existed as a mixed population of cells with distinct patterns of gene methylation. This result indicates that subclones of Huh-7 cells become highly permissive for HCV replication by having their antiviral genes epigenetically silenced through DNA hypermethylation. In addition to driving transcription of genes encoding inhibitors of cell cycle, activation of CREB3L1 also induces the expression of genes involved in assembly of collagen matrix. Transforming growth factor-β (TGF-β) is well known to induce excessive synthesis of collagen causing tissue fibrosis. We found that sustained induction of collagen synthesis by TGF-β required proteolytic activation of CREB3L1. RIP of CREB3L1 was inhibited by transmembrane 4 L6 family member 20 (TM4SF20), which retained CREB3L1 in the endoplasmic reticulum thereby separating CREB3L1 from the Golgi-resident proteases catalyzing the RIP reaction. TGF-β inhibited TM4SF20 expression through activation of extracellular signal-regulated kinases to stimulate RIP of CREB3L1. This cleavage allowed the NH2-terminal fragment of CREB3L1 to enter the nucleus where it forms a complex with Smad4 to activate transcription of genes involved in assembly of collagen extracellular matrix. Our study suggests that RIP of CREB3L1 could be a drug target to treat tissue fibrosis. Doxorubicin, a drug used in chemotherapy, is found to inhibit cell proliferation by inducing synthesis of ceramide, which in turn activates RIP of CREB3L1. Yet the mechanism through which ceramide activates CREB3L1 is not clear. Here we reported that ceramide induced alternative translocation of TM4SF20: In the absence of ceramide, the NH2-terminus of TM4SF20 was used as signal sequence to insert the polytopic membrane protein into ER. Upon treatment of ceramide, the NH2-terminus of TM4SF20 was no longer recognized as signal sequence. Instead, the NH2-terminus of newly synthesized TM4SF20 proteins was located in the cytosol, resulting in alternative translocation of the protein that adopted a membrane topology opposite to that in the absence of the lipid. We further demonstrated that insertion of the NH2-terminal sequence of TM4SF20 into ER in the absence of ceramide required translocation associated membrane protein 2 (TRAM2), as knockdown of TRAM2 led to alternative translocation of TM4SF20 even in the absence of ceramide. Since TRAM2 contains a domain known to bind ceramide, we suspect that ceramide may inhibit the function of TRAM2. These results suggest that ceramide could induce RIP of CREB3L1 by inactivating TM4SF20 through alternative translocation.Item Spectrum of autoinflammatory interferonopathies with features of vasculitis(2022-10-07) Shwin, Kyawt