Intercepting Cyclic Dinucleotide Signaling with Small Molecules

Bacterial infections, especially the ones that are caused by multidrug-resistant strains, are becoming increasingly difficult to treat and put enormous stress on healthcare systems. Recently President Obama announced a new initiative to combat the growing problem of antibiotic resistance. New types of antibiotic drugs are always in need to catch up with the rapid speed of bacterial drug-resistance acquisition. Bacterial second messengers, cyclic dinucleotides, play important roles in signal transduction and therefore are currently generating great buzz in the microbiology community because it is believed that small molecules that inhibit cyclic dinucleotide signaling could become next-generation antibacterial agents. The first identified cyclic dinucleotide, c-di-GMP, has now been shown to regulate a large number of processes, such as virulence, biofilm formation, cell cycle, quorum sensing, etc. Recently, another cyclic dinucleotide, c-di-AMP, has emerged as a regulator of key processes in Gram-positive and mycobacteria. C-di-AMP is now known to regulate DNA damage sensing, fatty acid synthesis, potassium ion transport, cell wall homeostasis and host type I interferon response induction. Due to the central roles that cyclic dinucleotides play in bacteria, we are interested in small molecules that intercept cyclic dinucleotide signaling with the hope that these molecules would help us learn more details about cyclic dinucleotide signaling or could be used to inhibit bacterial viability or virulence. This dissertation documents the development of several small molecule inhibitors of a cyclic dinucleotide synthase (DisA from B. subtilis) and phosphodiesterases (RocR from P. aeruginosa and CdnP from M. tuberculosis). We also demonstrate that an inhibitor of RocR PDE can inhibit bacterial swarming motility, which is a virulence factor.

Alternate Conformations Regulate Ribosomal Recoding in a Positive-sense RNA Virus

Positive-sense RNA viruses are important animal, plant, insect and bacteria pathogens and constitute the largest group of RNA viruses. Due to the relatively small size of their genomes, these viruses have evolved a variety of non-canonical translation mechanisms to optimize coding capacity expanding their proteome diversity. One such strategy is codon redefinition or recoding. First described in viruses, recoding is a programmed translation event in which codon alterations are context dependent. Recoding takes place in a subset of messenger RNA (mRNAs) with some products reflecting new, and some reflecting standard, meanings. The ratio between the two is both critical and highly regulated. While a variety of recoding mechanisms have been documented, (ribosome shunting, stop-carry on, termination-reinitiation, and translational bypassing), the two most extensively employed by RNA viruses are Programmed Ribosomal Frameshifting (PRF) and Programmed Ribosomal Readthrough (PRT). While both PRT and PRF subvert normal decoding for expression of C-terminal extension products, the former involves an alteration of reading frame, and the latter requires decoding of a non-sense codon. Both processes occur at a low but defined frequency, and both require Recoding Stimulatory Elements (RSE) for regulation and optimum functionality. These stimulatory signals can be embedded in the RNA in the form of sequence or secondary structure, or trans-acting factors outside the mRNA such as proteins or micro RNAs (miRNA). Despite 40+ years of study, the precise mechanisms by which viral RSE mediate ribosome recoding for the synthesis of their proteins, or how the ratio of these products is maintained, is poorly defined. This study reveals that in addition to a long distance RNA:RNA interaction, three alternate conformations and a phylogenetically conserved pseudoknot regulate PRT in the carmovirus Turnip crinkle virus (TCV).