Browsing by Subject "Cyclic GMP"
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Item Heme-Based Oxygen Sensors of Commensal, Symbiotic, and Pathogenic Bacteria(2012-08-15) Tuckerman, Jason Robert; Gilles-Gonzalez, Marie-AldaDirect oxygen sensors are proteins that serve as "on-off switches" to cause reversible and adaptive changes in the activities of other proteins or genes, with great specificity in response to fluctuations in oxygen concentration. The heme-based oxygen sensors are a large class of direct oxygen sensors that feature direct binding of oxygen to a sensory heme-containing domain. This heme-binding region couples to a regulatory domain within the same polypeptide. The types of functionalities controlled by these oxygen-specific switches are diverse, and include the regulation of protein activities, gene expression, and second messenger elaboration. A primary focus of this work was the biochemical characterization of a pair of heme-based oxygen sensors involved in the control of the bacterial second messenger cyclic diguanylic acid (c-di-GMP) in Escherichia coli. We discovered that these enzymes, designated DosC and DosP, serve as a diguanylate cyclase and c-di-GMP phosphodiesterase pair that associate with components of the E. coli RNA degradosome in vivo. Importantly, one member of these degradosomes, PNPase, is a direct, high-affinity target of c-di-GMP. These findings imply that specialized oxygen-sensing degradosomes exist in E. coli. In these oxygen-sensing degradosomes cellular oxygen levels regulate PNPase processing of specific RNA transcripts via c-di-GMP. A secondary focus of this work was the characterization of a novel two-component system in M. tuberculosis involved in the non-replicating persistent phase of this bacterium in a typical TB infection. Here, the activities of two heme-containing histidine kinases, DosT and DevS, were discovered to be inhibited specifically by oxygen. As DosT and DevS are the primary regulators of the dormancy survival regulator (DosR/DevR) transcription factor, these results contributed a molecular explanation for the numerous observations linking oxygen and DevR to the dormancy phenotype of M. tuberculosis seen both in vitro and in vivo.Item Innate Immune Sensing and Signaling of Cytosolic DNA(2015-04-10) Wu, Jiaxi; Mendell, Joshua T.; Chen, Zhijian J.; Olson, Eric N.; Cobb, Melanie H.In eukaryotic cells, DNA is normally confined within the nucleus and mitochondria. DNA exposed in the cytosol is a danger signal that warns the host of invading microbial pathogens and triggers innate immune responses including the production of type-I interferons (IFNs). Endogenous DNA that is inappropriately cleared can also accumulate in cytosol and drive pathological inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). It is well known that cytosolic DNA induces IFNs through the STING-TBK1-IRF3 axis. However, how DNA is sensed in the cytosol and how this sensing event leads to the activation of STING remains elusive. Using a cell-free complementation assay, we identified cyclic GMP-AMP (cGAMP), as a novel eukaryotic second messenger generated by DNA stimulated or DNA virus infected cells. cGAMP contains a unique phosphodiester linkage combination (both 2'-5' and 3'-5'), for which we referred to it as 2'3'-cGAMP. 2'3'-cGAMP bound to STING with nanomolar affinity and induced a dramatic conformational change that led to its activation. Through biochemical purification and quantitative mass spectrometry, we identified the enzyme that synthesizes cGAMP in a DNA dependent manner. This enzyme, which we named cyclic GMP-AMP synthase (cGAS), turned out to be the long sought-after cytosolic DNA sensor. Structural and functional studies revealed that cGAS is activated by DNA-induced dimerization. We further generated and characterized a cGAS knockout mouse strain, which failed to produce IFNs and other cytokines in response to DNA stimulation and was more vulnerable to lethal infection by DNA viruses. Together, these results not only elucidate the mechanism of cytosolic DNA sensing, but also uncover a novel second messenger-mediated signaling mechanism in innate immunity.Item Toward the Rational Design of Better Antivirals: The Development of cGAMP as an HIV-1 Anti-Retroviral and the Genetic Surveillance of WNV Evolution(2017-04-17) Aroh, Chukwuemika Nnabuike; Pasare, Chandrashekar; Wakeland, Edward K.; Yan, Nan; Schoggins, John W.; Pfeiffer, Julie K.The innate immune response is the first line of defense against pathogens and thus represents the first hurdle viruses must overcome to cause severe disease in humans. Understanding the consequences of viral evolution can give insights to mechanisms of viral pathogenesis as well as the development of novel therapeutics. Here I studied two clinically important viruses: Human Immunodeficiency Virus (HIV) and West Nile Virus (WNV). HIV-1 has evolved several mechanisms to evade immune detection by the cGAS-STING cytosolic DNA sensing pathway. A small cyclic di-nucleotide, cGAMP, activates the same pathway by directly binding STING. Treatment with cGAMP, delivered by ultra-pH sensitive nanoparticles or by liposomes, in human peripheral blood mononuclear cells (PBMCs) induced potent and long-acting protection against replication of several laboratory-adapted and clinical HIV-1 isolates in contrast to the short-lasting effect of current anti-retroviral therapy (ARTs). These results present the first evidence for potentially developing cGAMP or other STING agonists as a long-acting antiretroviral immunotherapy. West Nile Virus (WNV) is a mosquito-borne Flavivirus which was introduced to North America in 1999 and is currently the leading cause of viral encephalitis. The lack of specific therapeutics or human vaccines makes WNV an ongoing public health threat. Now endemic, WNV is steadily evolving, but the contribution of positively-selected mutations to human disease remains unclear. In 2012 the second largest outbreak of human West Nile disease occurred in the U.S., with one-third of the cases happening in Texas. The outbreak was associated with groups of WNV carrying positively-selected mutations. By sequencing WNV in Texas from 2012-2015, we show that positively-selected mutations in WNV mediate increased circulation and over-wintering in the environment, which may promote increases of human disease. Additionally, we show evidence that the WNV population is still evolving new alleles. These results advance our understanding of the impact of WNV evolution to human disease, and may afford insights to the evolution of other invading flaviviruses, such as Dengue and Zika virus. Altogether, these results show that understanding the consequences of viral evolution can be harnessed towards overcoming challenges to the development of more effective therapeutics.