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UNRAVELING CYCLIC DINUCLEOTIDE SIGNALING IN IMMUNE CELLS AND DISCOVERY OF NOVEL ANTIBACTERIAL AGENTS
Cyclic dinucleotides (CDNs) such as the bacterial CDNs (cyclic-di-AMP, cyclic-di-GMP and 3’3’cyclic GMP-AMP) and mammalian CDN, 2’3’-cGAMP, are essential immune response second messenger signaling molecules. These CDNs act via Stimulator of interferon genes (STING)-TANK Binding Kinase 1 (TBK1)-Interferon Regulatory Factor 3 (IRF3) pathway. However, data from our lab and others indicate that beyond the classical STING-TBK1-IRF3 pathway, CDNs also regulate other signaling axes related to both inflammatory and non-inflammatory pathways. But, a global view of how these CDNs affect signaling in diverse cells or through non-STING pathways is lacking. There is also paucity of data on CDN modulated kinases and no global assessment of phosphorylation events that follow cyclic GMP AMP synthase (cGAS)-STING axis stimulation in immune cells. Herein, I have used a proteomics approach to determine signaling pathways regulated by bacterial CDNs, c-di-GMP and c-di-AMP in human gingival fibroblasts such as pathways related to nucleotide excision repair (NER) which ordinarily do not channel through STING (Chapter 3). Additionally, with the use of phosphoproteomics and bioinformatics, this project accomplished a system-wide phosphorylation analyses of T cells treated with 2’3’cGAMP and showed that 2’3’cGAMP impact various, yet unreported critical kinases (E.g. LCK, ZAP70, ARG2) and signaling pathways important for T cell function (Chapter 4). Asides known interferon signaling, these differentially phosphorylated kinases were involved in T cell receptor (TCR) signaling, myeloid cell differentiation, cell cycle regulation, and regulation of double strand break repair.
Another area of interest addressed by this project is the discovery of novel antibacterial agents against multi-drug resistant (MDR) bacteria. Thus, in Chapters 5 and 6, I show the identification, antibacterial activity and characterization of HSD1835 and HSD1919 as novel SF5 and SCF-containing membrane active compounds, highly potent against preformed MDR biofilms with fast bactericidal activity against persister bacteria. Plus, an exciting addition to the fight against MDR bacteria in Chapter 7, the discovery of HSD1624 and analogs, which are able to re-sensitize MDR and colistin resistant bacteria such as Pseudomonas aeruginosa from a colistin MIC of 1024 μg/mL to 0.03 μg/mL (64000-fold reduction). Ultimately, these compounds could be translated into anti-biofilm and, anti-MDR bacteria therapeutics, preventing repeated surgeries due to infections, and saving lives.